Mass spectrometric immunoassay

ABSTRACT

Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

INTRODUCTION

[0001] The present invention relates to a new and useful immunoassay andmore specifically to new and improved mass spectrometric immunoassayprocesses for the detection and/or quantification of one or moreantigens or antibodies in a single determining immunoassay. Hereinafter,singular terms are intended to include plural.

[0002] Financial Assistance for some of the work reported herein wasprovided by the United States Department of Energy under Grant numberDEFG02-91ER61127. The United States government may have certain rightsto this invention.

BACKGROUND OF THE INVENTION

[0003] Immunoassay techniques first came into wide usage with thedevelopment of radioimmunoassay (RIA) in which the specificity ofantigen-antibody binding was coupled with the high sensitivity ofnuclear particle detection to detect and quantify antibody-antigenbinding in the presence of a large background of non-specific material.Later, enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay(ELISA) immunoassays coupled the specificity of antigen-antibody bindingwith the sensitivity of enzyme chemical reactions to detect and quantifyan antigen-antibody binding by producing colored, fluorescent, bio- orchemiluminescent chromophore. EIA and ELISA exhibited an amplificationfactor as high as 10⁸, allowing sensitivities competitive with RIAwithout the disadvantages of radioactivity.

[0004] Typical ELISA diagnostics relied on an antigen having at leastone epitope to which an enzyme-linked antibody could bind with a highaffinity. An antigen was affinity-isolated from its biological systemand allowed to interact with the enzyme-linked antibody. The enzyme ofchoice was generally alkaline phosphatase or horseradish peroxidase,both of which generated a colored product upon digestion of appropriatesubstrates. Although detection of attomole levels of an enzyme has beendemonstrated, so that it was, in principle, possible to detect attomolelevels of an antigen, traditional immunoassays did not operate at thatlevel of detection because all were limited by non-specific binding ofthe enzyme-linked antibody to surfaces in the reaction well or vial.This produced a background response which restricted the detection limitof the technique which could not be discriminated against becausedetection was indirect.

[0005] A further limitation of the traditional immunoassays employingoptical detection was caused by the limited number of clearly resolvablecolored enzyme products, at most two or three, which limited thepossibility for an immunoassay to screen for multiple antigens in asingle sample. Multiple antigen immunoassays usually focused on a numberof separate immunoassays in an array of well plates each requiring itsown sample which clearly reduced the utility of this approach. The idealmulti-antigen immunoassay would detect a large number of discreteantigens with high specificity in a single specimen, would cover a largedynamic range, be quantifiable over that range, and could be performedrapidly, that is, in minutes rather than hours, for critical clinicalsituations and high general throughput.

[0006] The sensitivity of EIA and ELISA relied on the specificity of theaffinants used to bind with the antigen or antibody being detected.Expensive and hard to produce monoclonal antibodies were usually thereagent of choice because the specificity of monoclonal antibodies isvery high. Polyclonal antibodies whose specificity is low could be usedin theory but were not a practical choice for a reagent becausepolyclonal antibodies bind with several species of antigens making thedetection of the resulting antibody-antigen complex less specific for asingle given antigen species.

[0007] Yet another restriction to EIA and ELISA is that they requiredthe antigen-antibody binding to reach an equilibrium for quantification,making the immunoassay take several hours to perform.

[0008] Until about 1988, mass spectrometry of proteins and peptides wasthought difficult or impossible. At that time Karas and Hillenkamp(Analytical Chemistry, vol. 60, pp. 2299-2301, 1988) demonstrated thatproteins could be ejected into the gas phase by embedding them into anorganic matrix which was then literally exploded using a pulsed laserbeam. This technique is commonly referred to as matrix-assisted laserdesorption/ionization (MALDI). When MALDI was coupled to atime-of-flight (TOF) mass spectrometer, a new field of biological massspectrometry was opened.

[0009] While the new MALDI techniques opened the field biomolecular massspectrometry, the mass spectrometric analysis of complex biologicalmaterials was not possible because of matrix overloading. Recently,Hutchens et al. (Hutchens, T. W. and Yip, T.), Rapid Communications inMass Spectrometry, vol 7, 1993, pp. 576-580.) demonstrated theutilization of affinity capture methods to quasi-purify proteins in aspecimen prior to MALDI mass spectrometry. By quasi-purifying thespecimen being assayed Hutchens et al. effectively overcame the primarylimitation of MALDI mass spectrometry, namely, the suppression of ionsignal due to overloading of the matrix. They named their technique“surface-enhanced affinity capture mass spectrometry (SEAC)”. Theyfurther demonstrated their technique by using single stranded DNA whichthey immobilized on the mass spectrometer probe tip to quasi-isolate theprotein lactoferrin from preterm infant urine.

[0010] More recently Hutchens, T. W. and Yip, T., in an internationalpatent application which was published Dec. 8, 1994 (WO 94/28418),described a method and apparatus for using affinity capture to improvemass spectrometric characterization of biomolecules.

[0011] Presently, there is no mass spectrometric immunoassay which iscapable of qualitatively and quantitatively determining the presence ofsingle or multiple antigen or antibody species in a specimen. It istoward the fulfillment of that need that the present invention isdirected.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention relates generally to the fields ofimmunoassay and mass spectrometry and more particularly to a new anduseful mass spectrometric immunoassay methodology for unequivocallydetecting and/or quantifying one or more antigens or antibodies from aspecimen within the limits of detection.

[0013] The present invention combines and exploits the specificity ofantibody-antigen binding and the ability of the mass spectrometer tounequivocally identify molecules in various qualitative and quantitativestrategies to analyze one or more antigens or antibodies in a specimenwithin the limit of detection. Both qualitative and quantificationstrategies utilize an antibody or antigen to capture and isolate anotherantigen or antibody, respectively, from its surroundings, and thereaftermass spectrometrically analyze the isolated antibody or antigen afterrelease from the capturing agent. The specificity of theantibody-antigen reaction coupled with the ability of the massspectrometer to separate and unequivocally identify the captured andisolated antibody or antigen by its mass-to-charge ratio from othermolecules that may accompany it lends two dimensions of specificity tothe present invention.

[0014] Because detection using mass spectrometry lends an addeddimension of unequivocal specificity, the mass spectrometric immunoassayunder the present invention is an improvement over existing immunoassaysin several ways. First, the present invention does not requiremonoclonal antibodies as a reagent, polyclonal antibodies produceequally reliable results. Second, radioactive materials are not requiredat all. Third, the presence of other substances not the subject of theimmunoassay do not interfere with either detecting or quantifying thetargeted antigen or antibody. Fourth, an accurate multiplex immunoassayclearly detecting and/or quantifying virtually any combination ofdifferent antigens or antibodies is possible.

[0015] In addition, the rapid ease by which the mass spectrometer candetect an antigen or antibody regardless of whether the antigen-antibodybinding has reached equilibrium grants to the present invention animmunoassay that produces results in a short period of time, minutesrather than hours.

[0016] An article by Nelson, R., et al., published in AnalyticalChemistry, vol. 67, pp 1153-1158, on or about Mar. 31, 1995, describescertain portions of the present invention in detail and is hereinincorporated by reference.

[0017] Accordingly it is a prime object of the present invention toprovide a new and improved mass spectrometric immunoassay fordetermining whether one or more designated antigens and/or antibodiesare present in a specimen.

[0018] A further object of the present invention is to provide a noveland unique immunoassay for determining what specific antigens and/orantibodies are present in a given specimen.

[0019] Another object of the present invention is to provide a new andimproved immunoassay for determining how much detected antigen orantibody is present in a specimen.

[0020] These and still further objects as shall hereinafter appear arereadily fulfilled by the present invention in a remarkably unexpectedmanner as will be readily discerned from the following detaileddescription of an exemplary embodiment thereof especially when read inconjunction with the accompanying drawing in which like parts bear likenumerals throughout the several views.

BRIEF DESCRIPTION OF THE DRAWING

[0021] In the drawing:

[0022]FIG. 1 is a depiction of the general scheme of a multiplex massspectrometric immunoassay under the present invention showing affinitycapture and isolation of two antigen analytes and a modified variant ofone of the antigen analytes, denoted with a star, and showing theresulting mass spectrum which distinctly resolves each antigen analyteand modified variant;

[0023]FIG. 2 is a mass spectrum resulting from the mass spectrometricimmunoassay of the present invention of a venom-laced human blood samplefor the antigen, myotoxin a, showing a distinct mass spectrometricresponse for singly charged myotoxin a at the mass/charge (m/z) ratiocorresponding to the molecular weight of myotoxin a at 4,822 Da, and asecond distinct response at the m/z ratio corresponding to 5,242 Da,which is the molecular weight of the modified variant H-myotoxin a, usedas an internal reference species.;

[0024]FIG. 3 is a mass spectrum resulting from mass spectrometricanalysis, without affinity capture and isolation, of a venom laced humanblood sample for the antigen, myotoxin a, showing no distinct massspectrometric response at the molecular weight of myotoxin a, but rathershowing a mass spectrometric response for singly charged hemoglobin Aand B at the mass/charge (m/z) ratios corresponding to their respectivemolecular weights of about 16,000 Da.;

[0025]FIG. 4 is a working curve relationship between the concentrationof myotoxin a in venom laced human blood samples and the magnitude ofmass spectrometric immunoassay responses for myotoxin a, normalized withthe modified variant, H-myotoxin a, and demonstrating the working curvequantification strategy for quantifying myotoxin a;

[0026]FIG. 5 is a mass spectrum resulting from the mass spectrometricimmunoassay of the present invention of a venom laced human blood samplecontaining four modified variants of myotoxin a, demonstrating thebargraph quantitative strategy for quantifying the antigen, myotoxin a;

[0027]FIG. 6 is a mass spectrum resulting from the mass spectrometricimmunoassay of the present invention of a venom laced human blood samplecontaining the modified variant, H-myotoxin a, demonstrating therelative limit signal quantitative strategy for quantifying myotoxin a;

[0028]FIG. 7 is a mass spectrum resulting from the multiplex massspectrometric immunoassay of the present invention of a venom lacedhuman blood sample for myotoxin a and Mojave toxin showing distinct massspectral signals for myotoxin a and Mojave toxin located at theirrespective molecular weights;

[0029]FIG. 8 results from the mass spectrometric immunoassay of thepresent invention of six preparation samples containing variousconcentrations of the antigen, α-1-acid glycoprotein (A1AG), and aconstant concentration of the internal reference species, human serumalbumin (HSA) and is a mass spectrum of one of these preparationsamples, showing distinct signals for A1Ag at m/z ˜37,000 and for HSA atm/z ˜67,000;

[0030]FIG. 9 is a working curve relationship between the concentrationof α-1-acid glycoprotein (A1AG) in a specimen and the magnitude of themass spectrometric response for α-1-acid glycoprotein, normalized withthe internal reference species, human serum albumin;

[0031]FIG. 10 results from the mass spectrometric immunoassay of thepresent invention of the rattlesnake toxin, myotoxin a, in human bloodcontaining the venom of the Mojave rattlesnake, and to which variousaliquots of purified myotoxin a have been added to allow quantificationby the standard addition strategy, and shows a mass spectrum of a samplein which the concentration of myotoxin a was increased by 1250 nM overthe intrinsic level;

[0032]FIG. 11 is the standard addition line defining the relationshipbetween added myotoxin a and the mass spectral responses for myotoxin a,normalized to the mass spectral response for another venom component,Mojave toxin, which is present in the blood sample at a constantconcentration and consequently may be used as an internal referencespecies;

[0033]FIG. 12 is a mass spectrum of a venom laced blood samplecontaining five modified variants of myotoxin a resulting from the massspectrometric immunoassay of the present invention in Example 7,demonstrating a bargraph quantification strategy;

[0034]FIG. 13 is a mass spectrum resulting from mass spectrometricanalysis of the preparation made of multiple antigen species in Example7 showing responses at various m/z ratios characteristic of the variousantigens; and

[0035]FIG. 14 is the mass spectrum of Example 7, showing a mass spectralresponse for the single antigen, α-cobratoxin, and demonstrating thethird qualitative mass spectrometric immunoassay strategy of the presentinvention for inferentially detecting the antibody to α-cobratoxin in aspecimen by capturing the antibody for α-cobratoxin and utilizing thisantibody in the affinity reagent.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0036] The present invention relates to a new mass spectrometricimmunoassay and more specifically to new and improved processes for thedetection and/or quantification of one or more antigens or antibodies ina single immunoassay.

[0037] The present invention can be utilized to detect and/or quantifysingle or multiple antigens or antibodies in a specimen. Single terms asused herein are meant to include plural.

[0038] In its simplest context, the present invention involves twophases: capture and isolation of an antibody analyte or antigen analyte,followed by mass spectrometric analysis of the captured antigen analyteor antibody analyte. The present invention can be used for qualitativeand/or quantitative purposes. That is, the present invention- can beused to determine whether a certain antibody or antigen is present in aspecimen (qualitative), and/or to measure the amount of the antigen orantibody present in a specimen (quantitative).

[0039] A more detailed description of the several facets of thisinvention appears hereafter utilizing the following lexicon.

[0040] “Affinant” is the antibody and/or antigen which is used to makethe affinity reagent. Usually, an affinity reagent to analyze forantigens will be made with one or several types of antibody. Similarly,an affinity reagent to analyze for antibodies will be made with one orseveral different antigens. However, all quantitative analyses in thepresent invention require the affinity reagent to capture at least twodifferent types of molecule, one of which is the analyte, the othermolecule acting as an internal reference species. Sometimes, the analytemay be an antibody and the internal reference species an antigen, orvice versa. Where this is so, it would be necessary for the affinityreagent to contain both an antibody and an antigen. For example, becausethe number of resolvable types of antibody is small, a protein that isnot an antibody may make a useful internal reference species for anantibody, because it has a resolvable mass and a specific antibody isreadily available for it. In such a case, the affinity reagent needs tocontain an immobilized antibody having a specific affinity for thatprotein, in addition to an immobilized antigen having a specificaffinity for the antibody analyte.

[0041] “Affinity” is the ability of a molecule to bind with anothermolecule having proper fitting conformation.

[0042] “Affinity reagent” is a type of antigen or antibody immobilizedto a solid substrate. Preferably, immobilization is obtained bycovalently linking the antigen or antibody to the solid substrate,although non-covalent linkage may be acceptable. The antibody or antigencan be directly linked to the solid substance surface, or can beindirectly attached to the solid substrate by linking the antibody orantigen to a coating on the solid substrate.

[0043] “Allergen” is a particulate material capable of stimulating anallergic response in susceptible individuals. Typically, allergensinclude dusts, pollens, molds, spores, and particles of insect andanimal detrita. Each type of material may carry on its surface, manydifferent kinds of proteins capable of stimulating an allergic response.The allergic response is characterized in the human body by theproduction of elevated levels of an immunoglobulin, IgE, molecularweight ˜170,000 Da, which is distinguishable from other immunoglobulinssuch as IgG which have a different molecular weight (IgG: ˜150,000 Da).

[0044] “Analyte” except as otherwise defined, is an antibody or antigenthat is captured, isolated and mass spectrometrically analyzed for thequalitative purpose of detecting whether a specimen contains a certainantibody or certain antigen. Depending on the qualitative methodemployed, the analyte may be the certain antibody or certain antigenbeing looked for in the specimen, or may be another antibody or antigenused to indirectly detect the certain antibody or certain antigen.

[0045] “Antibody” is a protein that specifically binds with anothermolecule that possesses one or more unique antigenic sites.

[0046] “Antigen” is any molecule having an antigenic site that can bindto an antibody. It is apparent that the categories of molecules that maybe considered antigens under this definition is broad, essentially anymolecule that can bind with an antibody qualifies as an antigenincluding an allergen.

[0047] “Counterpart” when used to describe an antibody or antigen, is anantibody or antigen which has a molecular weight indistinguishable inthe mass spectrum from that of the analyte, and which gives a responseidentical to that of the analyte when both are subjected to massspectrometric immunoassay from specimens of identical concentration.

[0048] “Disassociation agent” is any active cause which disassociates orunbinds a captured analyte from the affinity reagent. A laserdesorption/ionization agent may also act as a disassociation agent.Examples of a disassociation agent include but are not limited to:addition of a buffer solution, heating, sonication, application of anelectric potential, or addition of a MALDI matrix.

[0049] “Effective amount” of affinity reagent is that amount necessaryto capture an adequate quantity of the analyte (and internal referencespecies where relevant) to achieve the desired result.

[0050] “Internal reference species” is an antibody or antigen whosespecific affinity for another antibody or antigen is exploited in themass spectrometric immunoassay for quantitative purposes. The internalreference species is captured, isolated and mass spectrometricallyanalyzed alongside the analyte.

[0051] “Laser desorption/ionization agent” is any active cause whichenables an analyte to be laser desorped/ionized. An example of a laserdesorption/ionization agent is the addition of a MALDI matrix.

[0052] “Mass spectrometrically analyzed” is analyzing the antigenanalyte or antibody analyte with a mass spectrometer resulting in a massspectrometric response. Generally, a mass spectrometer is an instrumentdesigned to determine the mass-to-charge ratio of ions. For thisprocess, it is necessary for the analyte molecules to be volatilized,and ionized. The ionized molecules are then accelerated by an electricfield into the analyzing device, which separates the ions by virtue of aproperty dependent on the mass-to-charge ratio (m/z). Among theseproperties are: deflection in a magnetic field, velocity afteracceleration through a fixed electric potential drop, cyclotron orbitalfrequency in a magnetic field, and trajectory in a radio frequencyquadrupole field. Because antigens and antibodies are usually relativelylarge molecules and cannot be volatilized under ordinary massspectrometric procedures, volatilization and ionization of the antibodyor antigen usually involves some kind of assistance. One commonprocedure of assistance is known as matrix-assisted laserdesorption/ionization, or MALDI. Therefore, “mass spectrometricanalysis” is intended to include techniques that assist the massspectrometer in volatilizing and ionizing the antibody or antigenmolecules when such assistance is needed.

[0053] “Mass spectrometric mixture” is a mixture containing an antibodyor antigen which can be laser desorped/ionized by a mass spectrometer.

[0054] “Mass spectrometric response” (or “Response”) is the responsereceived from a mass spectrometer indicating whether or when an ionstruck the mass spectrometer's detector and whether or when no ionstruck the detector. A non-zero response at a given mass-to-charge ratioindicates that the material mass spectrometrically analyzed contained agiven molecule that gave rise to the ion corresponding to the responseat that charge-to mass ratio. It is apparent that a zero massspectrometric response would indicate that the material massspectrometrically analyzed either did not contain that molecule, orcontained that molecule in levels below the limit of detection.

[0055] It is important to realize that the mass spectrometric responseis defined as a change in the level of the signal measured at the massspectrometer detector, not simply the absolute level of that signal. Insome types of mass spectrometer, and particularly in matrix-assistedlaser desorption/ionization time-of-flight mass spectrometers, there isa detectable signal, resulting in a non-zero baseline, throughout themass spectrum arising from such causes as background noise and thearrival of ions at the detector at times non-specific to theirmass/charge ratio. Thus, the mass spectrometric response for an ionspecies at a specific mass/charge ratio is defined as the change in themass spectrometer signal above this baseline signal level.

[0056] Detection of an ion at a given setting of electric and magneticfield strengths, or at a given time after the ion is accelerated, iscontrolled by the mass-to-charge (mass/charge) ratio of that ion therebyproviding an accurate means of identifying the molecule that gave riseto that ion by the molecule's mass-to-charge ratio. The number of ionsthat strike the detector at a given setting, or at a given time, isindicated by the magnitude of the mass spectrometric response. A “massspectrum” is a collection of mass spectrometric responses over a rangeof mass-to-charge ratios.

[0057] A mass spectrum is commonly expressed graphically in terms ofrelative intensity against mass-to-charge ratio (m/z). Where an ionarises from a molecule present in the material being massspectrometrically analyzed at or above the limit of detection, theresponse is expressed as mass spectral signals or peaks (signal, orpeak) at the relative mass-to-charge ratio of the ion. Where the ioncharge is +1, the mass-to-charge ratio for that ion is equivalent to themolecular weight of the ion. Molecular weight is expressed in Daltons(Da.) and may be used interchangeably with the term mass-to-charge ratiofor singly charged ions.

[0058] The magnitude of a mass spectrometric response can be measured interms of “intensity”, height of the response, or “integral”, the areaunder the response. It is apparent that mass spectrometric response canbe expressed in a variety of tangible and intangible forms including butnot limited to signals, charts, electronic data and electric currents. Azero response, a response without magnitude, at a given mass-to-chargeratio indicates the material being mass spectrometrically analyzedeither did not contain a molecule that could give rise to an ion of thegiven mass-to-charge ratio, or did not contain that molecule at a levelat or above the level of detection.

[0059] “Modified variant” is an internal reference species which is madefrom an antibody or antigen analyte or a counterpart thereof, hasaffinity for the same antigen or antibody as does the analyte, andproduces a mass spectrometric response relative to the analyte which isfixed and readily determinable, but has a different molecular weightsuch that it is resolvable in the mass spectrum from the analyte.

[0060] “Non-specific affinity” is the strongly attractive interactionwith a broad range of antibody or antigen species. By way of exampleonly, the substance Protein A has a strong affinity for all antibodiesof the IgG family.

[0061] “Normalize” or any form of the word, is that procedure forcorrecting mass spectra for differences in instrument performance duringmass spectrometric immunoassay of multiple analytical systems. Inessence, normalization provides for accurate results when twoimmunoassays are compared in the quantitative strategies of the presentinvention. Normalization is preferably achieved with the assistance ofan internal reference species, but conceivably can be achieved withoutthe assistance of an internal reference species if instrument parametersare carefully controlled so as to be identical in the mass spectrometricimmunoassay of multiple analytical systems. Alternatively, if thedependence on the instrument parameters of the mass spectrometricresponse to a given analyte concentration is sufficiently well known,the mass spectrometric response may be corrected for the effects ofchanging instrument parameters.

[0062] “Post-combination affinity reagent” is an affinity reagent whichhas been allowed to bind with its target antigen or antibody.

[0063] “Preparation” is an operator created substance or material andused for a particular purpose in the mass spectrometric immunoassay. Theterm is used to distinguish it from the specimen. Under one method ofthe present invention, material containing a counterpart antigen iscreated for the purpose of determining whether a certain antibodyspecies is present in a specimen, such as antiserum, and is termed the“preparation” to distinguish it from the specimen and avoid confusion indescribing the method. Under several quantitative methods, a materialcontaining a counterpart antigen or antibody is created and termed the“preparation” and used in the quantification methods.

[0064] “Solid substrate” is defined as any physically separable solid towhich an antibody or antigen can be directly or indirectly attachedincluding but not limited to agarose beads, nylon metals, glass,silicon, and organic membranes.

[0065] “Specific affinity” is the strongly attractive interactionbetween specific antigens and their corresponding antibodies. Theselective attraction between an antigen and its corresponding antibodyoccurs between the antigenic site, or epitope, of the antigen and arecognition region in the antibody. As such, it may be possible tomodify an antigen by altering its molecular weight, without noticeablyaltering the epitope, so that the modified antigen also has a specificaffinity for the same antibody as the unmodified antigen. An antibodymay similarly be modified without eliminating its specific affinity forits antigen. The high specific affinity between antigens and antibodiesis the key to the selective capture and isolation of a specified antigenor antibody in an immunoassay.

[0066] “Specimen” is any material which is the focus of the massspectrometric immunoassay. Frequently the specimen will be of biologicalorigin, for example a blood sample, and contain analytes that aresimilarly of biological origin, for example peptides, proteins andantibodies. However, it is possible that the specimen may benon-biological in origin, for example a groundwater specimen, and theanalyte similarly may be of non-biological origin, for example apesticide. All that is necessary for a successful immunoassay is that itshould be possible to prepare an antibody having a specific affinity forthat analyte.

[0067] “Unbound remainder” is whatever is left after the affinityreagent screens the specimen.

[0068] The “mass spectrometrically immunoassay” is that procedure inwhich a specimen is incubated with an affinity reagent which willspecifically capture an analyte, for a time sufficient for the affinityreagent to bind a detectable fraction of the analyte, creating apost-combination affinity reagent. The post-combination affinity reagentis then separated from the unbound remainder of the specimen sample toisolate the captured antigen from the unbound remainder. Preferably, theisolated post combination affinity reagent is then washed to remove anyunbound remainder adhering to the isolated affinity reagent.

[0069] A laser desorption/ionization agent is then added to isolatedpost combination affinity reagent to unbind any antigen bound to theaffinant and form a mass spectrometric mixture from which the unboundantigen is then mass spectrometrically analyzed. If the specimencontained the certain antigen at a level above the detection limit ofthe mass spectrometric immunoassay process for that antigen, theresulting mass spectrum will show a mass spectral signal at the uniquemass-to-charge ratio of the certain antigen. If the specimen did notcontain the certain antigen at a level above the detection limit of themass spectrometric immunoassay process for that antigen, no such signalwill be evident.

[0070] Such a mass spectrometric immunoassay procedure can result in aqualitative or a quantitative analysis, depending on whether or not aquantification method is used.

[0071] A multitude of qualitative and quantitative methods are possibleusing the present invention. Below three qualitative methods arediscussed in detail, followed by a detailed discussion of sixquantification methods. The mass spectrometric immunoassay of thepresent invention is very sensitive and requires only very smallquantities of analyte and/or small volumes of analyte containingsamples. The detection limit is approximately between 1×10⁹ and 1×10¹⁰analyte molecules in about 200 μL (microliters) of sample.

[0072] The first qualitative strategy is designed to determine whetherone or more antigens are present in or are absent from a specimen.Antibodies to each antigen to be detected are immobilized on a solidsubstrate creating the affinity reagent. The affinity reagent is thenincubated with the specimen to screen the specimen for any antigenpresent in the specimen that has an affinity for the affinant-antibodyimmobilized on the affinity reagent. Screening in this context means tocombine the specimen and affinity reagent for enough time to allow theaffinant-antibody to combine with a detectable amount of antigen forwhich it has a specific affinity. The affinity reagent will capture someor all of the antigen present in the specimen, if any. Thepost-combination affinity reagent is then separated from the unboundremainder of the specimen to isolate the captured antigen from theunbound remainder. Preferably, the isolated post-combination affinityreagent is then washed to remove any unbound remainder adhering to theisolated affinity reagent.

[0073] A disassociation agent is applied to the post-combinationaffinity reagent to disrupt the antibody-antigen binding and produce asolution containing free antigen which can be separated from the solidaffinity reagent by filtering. If the mass spectrometric analysis is tobe performed using matrix-assisted laser desorption/ionization (MALDI),the sample must be combined with an excess amount of a laserdesorption/ionization agent (known otherwise as a “MALDI matrix”) andallowed to crystallize by drying. In general the MALDI matrix materialswhich are effective laser desorption/ionization agents are alsoremarkably effective disassociation agents, and disassociation usingthese materials is preferred, in particular because the process ofdisassociation and creation of a mass spectrometric mixture is therebymade simple and rapid.

[0074] The solution resulting from the disassociation step may be massspectrometrically analyzed. If the solution is to be analyzed usingmatrix-assisted laser desorption/ionization mass spectrometry, it mustfirst be combined with a MALDI matrix if such a matrix material was notused as a disassociation agent. If the specimen contained one or more ofthe antigen species for which the affinity reagent has a specificaffinity, at a level above the detection limit of the mass spectrometricimmunoassay process for that antigen, the resulting mass spectrum willshow a mass spectral signal at the unique mass-to-charge ratio of thecertain antigen. If the specimen did not contain the certain antigen ata level above the detection limit of the mass spectrometric immunoassayprocess for that antigen, no such signal will be evident. This strategymay be used to screen specimens for as many discrete antigen species ascan be distinguished from each other in a single mass spectrum. In theevent that more antigens must be searched for than can be distinguishedin a single mass spectrum, two or more affinity reagents must becreated, each having affinities to different sets of distinguishableantigens.

[0075] The procedures for the second qualitative strategy under thepresent invention are similar to the first except the affinity reagentis made with antigens seeking antibodies. The analyte is an antibody.The same general protocols apply. An antigen known to have a specificaffinity for the antibody analyte is immobilized to a solid subtrate.The affinity reagent is incubated with the specimen being assayed forthe desired antibody and allowed to capture the antibody for which ithas a specific affinity. After washing the reagent any captured antibodyis isolated from the specimen environment. A disassociation agent then alaser desorption/ionization is added to the isolated antibody to free itfrom the substrate and facilitate ionization prior to mass spectrometricanalysis. Alternately, only the laser desorption/ionization agent can beadded without the disassociation agent to the isolated antibody analyte.A mass spectral response at the molecular weight (same as single chargedm/z) of the target antibody evidences the presence of the antibody inthe specimen.

[0076] In addition to using pure antigens to capture specificantibodies, it can also be useful to use impure antigens to captureclasses of antibodies. Such a situation arises in testing for allergies,in whiich whole allergen particles, such as dusts, pollens, molds,spores and particles of insect and animal detrita, may be covalentlyanchored to a solid substrate, or even used without such anchoring ifthe particles are sufficiently large to be separated by filtering may beused to search for elevated levels of an antibody, IgE, which isproduced as part of the allergic response. IgE has a molecular weight of˜170,000 Da, sufficiently different from that of IgG, another majorantigenic antibody class with a molecular weight ˜150,000 Da, that IgEand IgG are distinguishable in a mass spectrometer. However, like otherantibodies, individual types of IgE molecules are not resolvable fromeach other. Accordingly, determination of which of several possibleallergens has a specific affinity for IgE in a blood sample wouldnecessarily use a single species of allergen, for example, the pollen ofa specific plant, such as ragweed, to screen a specimen for IgEcharacteristic of allergy to ragweed pollen, and separate screeningprocedures would be carried out for each specific allergen. Smallmolecules, such as drugs, do not stimulate an allergic response whenpresent alone, but do so effectively when bound to proteins to formconjugates. Screening for life-threatening sensitivity to drugstherefore would necessarily use affinity reagents in which the drugmolecules in question were attached to proteins incorporated in theaffinity reagent.

[0077] The advantage of such screening over the conventional skin andpatch tests for allergy is not only its enhanced speed but, equallyimportant, the ability to avoid a potentially catastrophichyperallergenic reaction that could occur when the patient is used as anallergy detector.

[0078] Because antibodies within a certain class, such as immunoglobulinG (IgG) do not vary in molecular weight by a sufficient amount to bedistinguishable mass spectrometrically, a different strategy must beadopted to screen a specimen for specific types of similar antibodies,for example, antibodies belonging to the IgG class. In this strategydetection of a specific antibody is indirect. A specimen is combinedwith a solid substrate having a broad non-specific affinity forantibodies. A representative sample of the antibody population in thespecimen will bind with the solid substrate creating an affinityreagent. The affinity reagent is then incubated with a screeningpreparation made with an antigen or antigens known to have a specificaffinity for the antibody or antibodies to be searched for. If a certainantibody is a member of the antibody population on the affinity reagent,and the specific antigen to that antibody is present in the screeningpreparation, then that antibody will capture its specific known antigenfrom the preparation. The affinity reagent is then separated from thepreparation leaving behind the unbound remainder. Preferably, theisolated affinity reagent is washed to remove any unbound remainderadhering thereto.

[0079] A laser desorption/ionization agent is then applied to theisolated affinity reagent to unbind any antigen bound to theaffinant-antibody forming a mass spectrometric mixture from which theunbound antigen is then mass spectrometrically analyzed. A mass spectralsignal at the unique mass-to-charge ratio of a known antigen indicatesthe antibody specific for that antigen was present in the specimen.Absence of a mass spectral signal indicates that the certain antibodywas not present in the specimen at or above the detectable limit.

[0080] The preferred incubation method for all three qualitative methodsis arrived at empirically and depends on the given analytical system.Generally, incubation can be stationary (combining the affinity reagentand sample then agitating), flowing (one-time or repetitive flowing ofsample over the affinity reagent), or accelerated by the application ofan electrical potential across the sample solution. Because the use ofan internal reference species serves to calibrate variables in theincubation process as well as in the mass spectrometric analysis, it isnot necessary to ensure that incubation proceeds to equilibrium toachieve accurate analyses. Rapid incubations and consequently rapidanalyses are therefore possible under the present invention.

[0081] Preferably, the disassociation agent and laserdesorption/ionization agent chosen is arrived at empirically, however,the preferred agent is often the addition of any material commonlyreferred to as a “MALDI matrix” by those in the field of massspectrometry. Examples of commonly used MALDI matrix materials can befound in: Fan Xiang and Ronald C. Beavis, Rapid Communications in MassSpectrometry, vol. 8, pp. 199-204, 1994 and in Ronald C. Beavis, OrganicMass Spectrometry, vol. 27, pp. 653-659, 1992.

[0082] Because an unbound antigen analyte or unbound antibody analyte istypically too large a molecule to be ionized, the unbound analyte cannotordinarily be mass spectrally analyzed without additional preparatorytechniques. Preferably, matrix assisted laser desorption/ionization(MALDI) techniques enable mass spectrometric analysis of the analyte.Standard MALDI protocols can be found in Ronald C. Beavis, Organic MassSpectrometry, vol. 27, pp. 653-659, 1992.

[0083] The mass spectrometer used in the present invention can be anymass spectrometer that will analyze the analytes including a magneticsector mass spectrometer, a Fourier transform ion cyclotron resonance(FTICR) mass spectrometer, a quadrupole ion trap mass spectrometer andany time-of-flight (TOF) mass spectrometer. Of these the TOF massspectrometer is preferred.

[0084] Mass spectrometric analysis of the captured, isolated and unboundanalyte results in an analyte mass spectral signal at the mass-to-chargeratio characteristic of the analyte. The location of the signal on themass spectrum is dependent on the molecular weight of the analyte,thereby providing a reliable means for identifying the analyte. The massspectral signal also has magnitude. The magnitude of the signal isindicative of the amount of analyte that is ionized and detected by themass spectrometer. Mass spectrometric signal magnitude has at least twodimensions that are directly measurable, intensity or height of thesignal, and integral or the area under the signal; Either the intensityor the integral can be used to quantify.

[0085] The mass spectrometric immunoassay process can also be designedto quantify an antibody or antigen present in a specimen. All suchquantitative analyses utilize standard preparations containing knownconcentrations of the analyte for calibration. In addition, because itis difficult to control the analytical conditions sufficiently well toensure a constant absolute mass spectrometric response for a constantanalyte concentration in different samples, quantitative analysis usingthe present invention relies on the presence of or the introduction ofat least one internal reference species in the analytical system priorto incubation with the affinity reagent. The internal reference speciescan be added to the analytical system being assayed, or be intrinsicthereto. It is captured, isolated and mass spectrometrically analyzedsimultaneously with the analyte, thereby serving to calibrate theanalytical conditions from one analysis to another because both analyteand internal reference species respond identically to changes in theseconditions.

[0086] The affinity reagent must contain an affinant that willspecifically capture or bind with the internal reference species. It isessential that the internal reference species have a molecular weightsufficiently different from that of the analyte or analytes that it canbe resolved in the mass spectrum from the signals arising from theanalyte or analytes. However, the molecular weight difference ispreferably the minimum necessary for resolution in the mass spectrum,because analytes differing very greatly in mass may not respondidentically to changes in the mass spectrometer operating conditions.Preferably, the internal reference species is a modified variant of theanalyte. Where a modified variant is used as the internal referencespecies, an affinant that can capture the analyte can usually alsocapture the modified variant because the modification shifts themolecular weight of the antibody or antigen without destroying itsaffinity. Where the internal reference species is a not a modifiedvariant of the analyte, another immunochemical affinity group must bepresent in the affinity reagent in order to simultaneously capture andisolate that internal reference species alongside the analyte. It ispossible, and may be desirable, for a protein that is not an antibody tobe used as an internal reference standard in an analysis of an antibodyspecies. In such a situation, the affinity reagent would be preparedwith two classes of molecules, an antibody specific for the protein andan antigen for which the analyte is specific.

[0087] The number of internal reference species employed also depends onthe quantification method employed. The working curve or single pointquantification approaches require at least one internal referencespecies present in the analytical system. The bargraph quantificationapproach requires several internal reference species.

[0088] The internal reference species in a quantification method servestwo roles: 1) the role of a normalizer, and 2) the role of aconcentration indicator. There is more than one possible strategy ormethod for quantification of an antigen or antibody under each of theseroles.

[0089] The internal reference species serves to correct for differencesin affinity reagent activity or concentration, for differences inincubation times for different samples, and for differences in massspectrometer performance during the mass spectrometric immunoassay ofmultiple samples, thereby ensuring accurate quantification. However,because all mass spectrometric responses are thereby determined relativeto the internal reference species response, this internal referencespecies signal also serves as a concentration indicator, that is, theinternal reference species signal for a given concentration of thatspecies is calibrated by various strategies so as to indicate a givenconcentration of the analyte species.

[0090] Quantification strategies all involve mass spectrometricimmunoassay of at least two separate analytical systems. In most of thequantitative strategies, one analytical system contains the specimen,and the other a preparation containing the antibody or antigen analyteor a counterpart thereof in known concentration, or in a concentrationwhich has been altered by a known amount by adding a known amount of theanalyte where both the specimen and the preparation contain the sameinternal reference species in known concentration, preferably the same.

[0091] Normalizing with an internal reference species can beaccomplished in two basic ways, that is, during mass spectrometricanalysis or after. Both methods require the analytical samples tocontain the same internal reference species. For normalizing during massspectrometric analyses, the mass spectrometer is adjusted so as to bringthe internal reference species signals of each immunoassay to a valueproportional to the concentration of the internal reference species.Most conveniently, the internal reference species concentrations in allthe analytical samples are the same, and the mass spectrometer isadjusted to bring all internal reference species signals to the samevalue. For normalizing after mass spectrometric analyses, the massspectrum for each assay is divided or multiplied by an appropriatefactor, again to bring the internal reference species mass spectralresponses to a value proportional to the concentration of the internalreference species. The second procedure is preferred. The result is tonormalize all mass spectral responses or signals, and all affinitycapture procedures to one standard thereby artificially, or in practice,making instrument performance and affinity capture procedures in each ofthe immunoassays the same. The working curve strategy, standard additionstrategy, single point calibration strategy, and the bargraph strategyare all quantification strategies under the present invention thatemploy one or more internal reference species as both a normalizer and aconcentration indicator.

[0092] Although it is preferred that the normalization of mass spectrabe achieved where the internal reference species concentration in eachanalytical sample being mass spectrometrically assayed is the same ineach analytical sample, it is possible to normalize multiple massspectra with analytical samples containing the same internal referencespecies in different, but known concentrations. To normalize under theseconditions a scale is determined for the magnitude of that internalreference species mass spectral response and that scale is used tonormalize the other mass spectra. By way of illustration only, supposesample A contains the internal reference species, I-A, in concentrationof 100, and sample B contains the same internal reference species,termed I-B to distinguish from A's internal reference species, inconcentration of 50. After mass spectrometric immunoassay of A and B,the mass spectral signal for I-A is adjusted to fit an intensity of one(normalized intensity), the other mass spectral signals in A's massspectrum are also adjusted so as to be in the same original proportion(in intensity) to the adjusted I-A signal as they were for the initialI-A signal. Because the concentration of I-B is half the concentrationof I-A, the mass spectral signal for I-B then adjusted to fit anormalized intensity of 0.5. The other mass spectral signals in B'sspectrum are also adjusted so as to be in the same original proportion(in intensity) to the adjusted I-B signal as they were for the initialI-B signal. Preferably, all spectra adjustments are accomplished withthe aid of computer data manipulation software such as LABCALC, producedby Galactic Industries, Salem, N.H.

[0093] For accurate normalization and/or calibration using an internalreference species the absolute amplitudes of the mass spectrometricsignals for the analyte and internal reference species should besimilar. If these two signals differ by a large factor, for example by afactor of several hundred, it will not be possible to measure bothsignals accurately in the same mass spectrum because the detectionsystem cannot respond accurately to signals differing by so large afactor, i.e., the dynamic range is limited. For example, in thetime-of-flight mass spectrometers which are the preferred devices forobtaining mass spectra of laser-desorped ions in the present invention,the signal from the mass spectrometer must typically be digitized, thatis, converted to a binary number suitable for transfer to a computermemory, at successive time intervals as short as 5-10 nanoseconds. Thedigitization circuitry typically converts the signal to a binary numberhaving a value between 0 and 256. If a 1 volt signal, for example,corresponded to the digital value of 256, then all signals larger than 1volt would also be registered as 256 and the system would have noability to measure any such signals accurately. Conversely, in theabsence of any electronic noise, all signals lower than 1/256 voltswould be registered as 0 and again the measurement would be inaccurate.Low signals above 1/256 volt are also measured inaccurately because thebinary scale is coarse and because of noise effects. Preferably, theabsolute amplitudes of the mass spectrometric signals for the analyteand internal reference species should differ by less than a factor often, and the mass spectrometer is adjusted to bring the more intensesignal near the allowable maximum.

[0094] The mass spectral signal of an internal reference species istypically used to directly quantify an analyte by comparing the internalreference species signal to the analyte signal, after the internalreference species signal has been calibrated by a working curve orstandard addition approach.

[0095] It is apparent that given the high specificity of affinitycapture and isolation, and of mass spectrometry, quantificationstrategies can be employed to quantify multiple antigens or antibodiesin the same specimen. It is also apparent that it is possible to employmultiple quantification strategies in a given assay to quantify singleor multiple antibodies or antigens.

DETAILED QUANTIFICATION METHODS USING THE FIRST AND SECOND QUALITATIVEMASS SPECTROMETRIC METHODS

[0096] Below is a detailed description of each of the quantificationstrategies as employed with the first and second qualitative massspectrometric immunoassay methods.

[0097] It is apparent that, because analytical parameters may vary,including differences in affinity reagent activity or concentration,differences in incubation times for different samples, differences inelution efficiency, and differences in mass spectrometer performanceduring the mass spectrometric immunoassay of multiple specimens,therefore the absolute intensity of the mass spectrometric response fora given analyte in a given analysis cannot be used to derive aconcentration for that analyte. Instead, an internal reference species(IRS), which is preferably captured by the same affinity reagent duringthe same incubation time, eluted under identical conditions and massspectrometrically analyzed under identical conditions, serves tocalibrate all of these variables at one time. All analyte signals aretherefore determined as ratios to the respective IRS signals. When thisis done it is possible also to calibrate the analysis by determining theanalyte/IRS signal ratio for a known concentration ratio of analyte andIRS in an analytical sample. The methods for accomplishing thiscalibration are known as the working curve approach, the standardaddition approach, and the single point calibration approach, and areused here exactly as in standard analytical chemistry procedures.

[0098] The working curve approach is developed in anticipation of thefact that generally the analyte concentration encountered in an analysiswill not be identical to the analyte concentration used to calibrate theIRS signal. There are two possible approaches.

[0099] a) It may be assumed that the relationship between analyte/IRSconcentration ratio and analyte/IRS signal ratio is accurately linear.Then, for example, if the analyte/IRS signal ratio is 2:1 for equalconcentrations of analyte and IRS, an analyte/IRS signal ratio of 4:1signals an analyte concentration exactly twice the known IRSconcentration in the sample. It is usually safe to assume that such alinear assumption may hold, with an accuracy of approximately 10% orbetter, for analyte/IRS concentration ratios between approximately ⅓ and3 times the ratio at which the original calibration was performed (i.e.,over about a factor of 10 in concentration). When this assumption oflinearity is valid, the relationship between analyte concentration andinternal reference mass spectrometric response may be determined using asingle calibration sample and this approach is known herein as thesingle point calibration method.

[0100] By way of illustration, suppose signal A for analyte A of knownconcentration 1000 has a magnitude of 10 units, and signal B for analyteB of unknown concentration has a magnitude of 7.2 units, and it is knownfrom prior calibration that equal concentrations of both analytes willyield equal mass spectral signals. In calibrating A's signal, each unitof A's magnitude represents a concentration of 1000/10 or 100. AnalyteB's concentration is therefore 7.2×100 or 720 units. If equalconcentrations of A and B yield different mass spectral responses thecalibration must include the ratio of the mass spectral responses. Forexample, if the mass spectral signal for a given concentration of A were½ the mass spectral signal for the same concentration of B, then theconcentration of B corresponding to a signal magnitude of 7.2 units inthe above example would be 720/2 units, or 360 units.

[0101] For simple ratio quantification of analytes in separatespecimens, the specimens (with internal reference species present) areeach mass spectrometrically immunoassayed with an affinity reagenthaving an affinity for each antigen or antibody being looked for in therelevant specimen, and for the internal reference species. The resultingmass spectra are normalized using the internal reference species signalsthen the signal of the analyte of unknown concentration is calibratedagainst the signal of the analyte of known concentration as described inthe hypothetical example above.

[0102] b) For situations where the analyte/IRS concentration ratio isexpected to vary over more than a factor of 10, or where therelationship is expected to be non-linear, or where greater accuracy isrequired, the relationship between analyte/IRS concentration ratio andanalyte/IRS signal ratio may be determined over as large a range asdesired by making a series of preparations with varying analyte/IRSconcentration ratios in which the analyte concentrations span theexpected analyte concentration range. The greater the number ofpreparations, the more accurately the relationship between analyte/IRSsignal ratio and analyte concentration will be determined. The antibodyor antigen analyte used to make the preparations is generically termedthe preparation analyte, or specifically termed the preparation antigenor preparation antibody. The preparations either contain the sameinternal reference species in the same concentration as the specimen, ora concentration known relative to that in the specimen. After subjectingeach sample to mass spectrometric immunoassay, the ratios of the analytemass spectrometric responses to the corresponding internal referencespecies mass spectrometric responses are plotted as a function of theknown ratios of the analyte/internal reference species concentrationsfor each sample. The resulting relationship, or working curve, may beexpressed in a variety of ways, including, but not limited to, a graph,a mathematical relationship or computerized data.

[0103] If the analyte (antigen or antibody) is present in the specimen,as evidenced by an analyte mass spectral signal on the specimen massspectrum, then the analyte can be quantified by locating the point onthe working curve relationship (ultimately a mathematical relationship)appertaining to the magnitude of the analyte/IRS signal ratio anddetermining the analyte concentration that corresponds to that point,given the known IRS concentration.

[0104] As an alternative to the generation of a working curve spanning awide range of analyte concentrations, over which the working curverelationship might be expected to be non-linear, or for extremely rapiddetermination of analyte concentrations directly from the mass spectrum,a unique bargraph quantification method may be used. The bargraphquantification method requires the presence of several distinct internalreference species in the analytical sample, each at a different andknown concentration and differing in mass from the certain antigen orantibody and each other sufficiently to be distinguishable in the massspectrum. Because the concentrations of the internal reference speciesmust be known, the internal reference species are typically added to thespecimen, not intrinsic thereto. Mass spectrometric immunoassay of thespecimen is then executed using an affinity reagent having specificaffinity for each internal reference species and for the certainantibody or certain antigen being looked for in the specimen. Theresulting mass spectrum will contain a mass spectral signal for eachinternal reference species and a mass spectral signal for the certainantigen or certain antibody, if present.

[0105] Preferably, the internal reference species are all modifiedvariants of the analyte being mass spectrometrically immunoassayed sothat the internal reference species are capable of being captured by asingle affinant, and the mass spectral signals all neighbor one anotheron the mass spectrum. Each distinct internal reference species must havea molecular weight sufficiently different from the other internalreference species so as to be resolvable in a single mass spectrum. Inaddition, it is preferable that the range of concentrations covered bythe internal reference species span the concentration range in which theanalyte is reasonably expected to be found. It is not necessary that thedifferent internal reference species be captured by the affinity reagentwith the same efficiency as either the analyte or each other; thedifferent internal reference species are each simply added to theanalytical sample at concentrations which produce mass spectral signalsthat have the same amplitude as the signal that would be produced by aknown concentration of the analyte.

[0106] With this bargraph mass spectrum, the analyte can be quantifiedin at least three ways: 1) by interpolating the analyte's mass spectralsignal magnitude to the magnitude of the internal reference species massspectral signal immediately above and below the analyte mass spectralsignal, normally assuming a linear relationship between signal andconcentration in this range, or 2) by estimating the analyteconcentration by simple visual comparison of the analyte mass spectralsignal and the nearest internal reference species signal (in magnitude)without computation, or 3) by certifying that the analyte signal, andtherefore the analyte concentration, is above or below the signal leveldue to a reference species corresponding to a previously determinedanalyte concentration. This previously determined analyte concentrationmay be chosen to specify the presence or absence of a specific diseaseor condition. Since, under the bargraph strategy, the analyte is thecertain antigen or certain antibody present in the specimen, thequantification of the analyte directly quantifies the certain antibodyor certain antigen. The advantage of the bargraph approach is apparentin that the calibration scale is built into the mass spectrum givingexceptional immunity to instrumental and sample variations, andextremely rapid direct readout. For the most accurate analysis strategy(1) above is used, and the accuracy of this approach may be improved asnecessary either by choosing the internal reference speciesconcentrations to produce mass spectral signals which are closely spacedand near the analyte mass spectral signal, or by calibrating the analyteconcentration range between those analyte concentration values specifiedby the internal reference species signals by using a number of standardpreparations containing analyte concentrations within the concentrationrange to be calibrated.

[0107] The standard addition approach is another strategy fordetermining the relationship between the analyte/IRS signal ratio andthe analyte/IRS concentration ratio. In this approach, separatecalibration preparations are not required. Instead the effect on theanalyte/IRS signal ratio of changing the concentration of the analyte inthe analytical sample by a known amount is determined. Most directly,the analytical sample, to which an internal reference species has beenadded, is divided into several divided samples, at a minimum two. Thefirst divided sample is mass spectrometrically immunoassayed and ananalyte/IRS signal ratio determined from the resulting addition-absentmass spectrum. To the other divided sample or samples various knownamounts of the analyte, or an analyte counterpart, are added to increasethe concentrations of the analyte, or the analyte counterpart, byvarious known amounts. These samples similarly are massspectrometrically immunoassayed resulting in a series ofaddition-present mass spectra from which the analyte/IRS signal ratiosare determined.

[0108] The analyte/IRS signal ratios in the addition-present massspectra are then used to determine the analyte concentration in theaddition-absent sample. This is preferably done by using theaddition-present analyte/IRS signal ratios closest in magnitude to theaddition-absent analyte/IRS signal ratio to establish a mathematicalrelationship between changes in magnitude of the analyte/IRS signalratio and the corresponding concentration change in the antibody orantigen due to the standard addition. This mathematical relationship maybe expressed in a variety of ways, including but not limited to a line,a mathematical function, a graph, or computerized data. The standardaddition mathematical relationship is then extrapolated to the interceptpoint for zero mass spectrometric response. The intercept point's valuefor standard addition concentration will be in negative units. Theabsolute value of this value inferentially represents the concentrationof the analyte in the specimen.

[0109] Less preferably, a standard addition analysis may be performed inserial fashion without dividing the original sample. In this approach,the analytical sample, to which an internal reference species has beenadded, is mass spectrometrically immunoassayed using an incubationprocedure designed to capture only a small fraction (for example, lessthan 5%) of the analyte and IRS, and an analyte/IRS signal ratiodetermined for this addition-absent sample. A known amount of analyte,or an analyte counterpart, is added to the original sample, increasingthe concentration of the analyte, or analyte counterpart, by a knownamount, and again the sample is mass spectrometrically immunoassayedusing an incubation procedure designed to capture only a small fraction(for example, less than 5%) of the analyte and IRS. An analyte/IRSsignal ratio is similarly determined for this addition-present sample.Further addition-present samples may be prepared by increasing theconcentration of the analyte or analyte counterpart by further knownamounts and these samples may be similarly mass spectrometricallyimmunoassayed to determine further analyte/IRS signal ratios.

[0110] The analyte/IRS signal ratios in the addition-present massspectral signals are then used to determine the analyte concentration inthe addition-absent sample exactly as in the parallel standard additionapproach. Since mass spectrometric immunoassay of each addition-presentsample serves to calibrate a sample in which the concentration of theanalyte differs from the analyte concentration in the addition-freesample by an amount which depends on the amounts of analyte captured inthe preceding mass spectrometric immunoassays, it is apparent that theaccuracy of this procedure will only be acceptable if the amount ofanalyte captured in each successive step is small, for example if 5% ofthe analyte is captured in the mass spectrometric immunoassay of theaddition-free sample and mass spectrometric immunoassay of a singleaddition-present sample is performed, the analyte concentrationdetermined thereby would be in error by 5%

[0111] The relative signal approach compares two analyte signals to eachother where the concentrations of the analytes in the specimens areunknown. Often times the precise concentration of an antibody or antigenis not the information needed. Rather, knowing whether the antibody orantigen is present in levels at, below, or above the level of anotherantibody or antigen is what is needed. It is irrelevant whether anumerical figure is reached because only the relative relationshipmatters. To compare two or more analyte concentrations to each other bymeans of their mass spectrometric immunoassay responses, it is necessaryonly to know the relative response of the analytical process to eachanalyte, i.e., the relative amplitude of the mass spectrometric signalswhen each analyte is present in a calibration sample at identicalconcentrations (or at a concentration ratio which is known). In thisstrategy, a separate internal reference species is not required becauseeach analyte serves to normalize the other, i.e., both analytes aresubjected to identical affinity capture procedures and to identical massspectrometric procedures.

[0112] The relative signal and simple ratio quantification methods sharesome similarities. Both compare the mass spectral signals of one analyteto another analyte. The two analytes can originate from one specimen,thus involving only one mass spectrum, or can each originate fromseparate specimens, thus involving two mass spectra. It is not necessarythat the analytes have identical molar responses, i.e., affinityconstants and desorption/ionization efficiencies.

[0113] It is apparent that either approach can be used for thequantification of more than two analytes and/or involve more than twospecimens. It is also apparent that the analytes from multiple samplesmay be the same type of analyte, or different. Therefore, the followingdescriptions are meant to include multiple specimens and/or multipleanalyte immunoassays.

[0114] When two or more specimens are involved, both the simple ratioand relative signal quantification methods require that an internalreference species be present in each specimen sample in the sameconcentration to normalize the mass spectra. If one specimen is used,signals for the two analytes must be distinguishable in the massspectrum, and no normalizing internal reference species is necessary. Itis apparent that the two analytes whose mass spectral signals are beingcompared in a single sample are used to quantify either or both of theanalytes and are essentially acting as analyte and an internal referencespecies, i.e., in qualitative and quantitative capacities at the sametime. Therefore, to avoid confusion of terms, “internal referencespecies” is used in connection with the relative signal and simple ratioquantification methods to mean the antibody or antigen occupying thenormalizer role. An “analyte” is used to mean the antigen or antibodyacting in both a qualitative and quantitative role.

[0115] For relative signal quantification of analytes in separatespecimens, the specimens (with internal reference species present) areeach mass spectrometrically immunoassayed with an affinity reagenthaving an affinity for each antigen or antibody being looked for in thatspecimen. The resulting spectra are normalized, then a comparison of theanalyte signals made to determine which analyte is of greater or lesserconcentration. By way of illustration only, suppose two blood samplesfrom the same person taken at different times are mass spectrometricallyimmunoassayed to determine whether there is a change in the level ofantigen X. Affinity reagent having an affinity for antigen X would beused in the mass spectrometric immunoassay of both samples. The two massspectra would each show a mass spectral signal for antigen X and theinternal reference species. After normalizing the spectra, the twoantigen X signals could be compared to see if the level of antigen X haddropped or risen in the intervening time between taking the samples. Theabsolute concentration of X in either sample can not be determinedwithout further calibration, but the amplitude of the relative changemay be determined.

[0116] In the simple ratio quantification strategy, two analyte massspectral signals are compared where one of the analyte signalsoriginates from a specimen where that analyte's concentration is known.Unlike the relative signal method above, a numerical quantificationresults. The unknown concentration analyte signal is calibrated againstthe known analyte mass spectral signal to determine the concentration ofthe unknown analyte in that specimen.

[0117] For simple ratio quantification of two analytes in the samespecimen, the specimen is mass spectrometrically immunoassayed with anaffinity reagent having an affinity for each antigen or antibody beinglooked for in that specimen. The resulting mass spectrum will contain asignal for each analyte which was present in the specimen. Where twosignals result, calibration of the magnitude of the signal correspondingto the analyte of known concentration to the magnitude of the signal ofthe analyte of unknown concentration determines the concentration of theother.

[0118] To further aid in the understanding of the present invention, andnot by way of limitation, the following examples are presented:

EXAMPLE 1

[0119] In one practice of the present invention, a single analyte,myotoxin a, was detected in human whole blood using the affinant,anti-myotoxin a, as a constituent of the affinity reagent, and thenquantified using a working curve strategy. Myotoxin a is one toxin foundin the venom of the prairie rattlesnake, Crotalus viridis viridis (C. v.viridis).

[0120] The antibody, anti-myotoxin a immunoglobulin IgG affinitypurified from rabbit antiserum, was used to prepare the affinity reagentas follows. One milliliter of antibody solution (the solution containedthe antibody at 5 mg/mL in 0.01M tris[Hydroxymethyl] aminomethane, pH8.2 (tris)) was incubated for two hours (gentle agitation at roomtemperature) with 1 mL of slurried 6% agarose beads on which protein Awas supported. Following incubation, the beads were washed (3×1 mL tris)and allowed to react at room temperature with 500 μL of 0.02 Mdimethylpimelimidate dihydrochloride prepared in 0.2 M triethanolamine(pH 8.2). The reaction was stopped after one hour by washing (3×1 mL)with 0.2 M triethanolamine followed by one milliliter 0.2 M sodiumcitrate buffer (pH 2.8). The reagent was finally washed (3×1 mL) withtris and resuspended in 500 μL of the same buffer.

[0121] Stock human blood (SB) was prepared after intravenous retrievalby immediately diluting to ten times volume with normal saline toprevent coagulation. Three specimens were prepared from this dilutedstock blood by combining 50 μL SB, 150 μL tris and 2 μL aliquot ofC.v.viridis venom at a concentration of 0.2 mg/Ml (resulting afterdilution in a concentration of 0.002 mg/mL venom).

[0122] An internal reference species for the quantification of myotoxina was prepared by lysine conversion of myotoxin a to homoarginine(H-myotoxin a). The lysine modification was carried out as follows:myotoxin a (final concentration=10 mg/mL) was dissolved in 0.5 MO-methylisourea, which was adjusted to pH 11.0 with 8.0 M NaOH. Thesolution was incubated at 4° C. for 120 hours. The reaction was stoppedby addition of an equal volume of 1 M sodium dihydrogen phosphate at pH5.0. The mixture was then desalted with an Amicon ultracentrifugationdevice fitted with a PM-2 membrane filter at 4° C. The resultingsolution was lyophilized and stored for future use in a desiccator at−20° C. The modification resulted in the conversion of each of the tenlysine residues to a guanidinium group, giving a total shift in mass of420 Da.

[0123] Three microliter aliquots of slurried affinity reagent wereincubated for 45 minutes with a 200 μL volume of the specimen containing0.002 mg/mL whole venom and 40 nM (nanomolar) H-myotoxin a. The affinityreagent, now containing myotoxin a affinity bound to the retainedanti-myotoxin a, was then physically separated from the specimen byforcing the entire 200 μL (microliter) volume through the backside of aP-10, 10 μL filter pipette tip thereby retaining the affinity reagent onthe filter. The affinity reagent was then washed by forcing rinses (2×1mL 0.1% Triton X-100, 2×1 mL tris, 1 mL triply-distilled water) throughthe P-10 tip.

[0124] After the final rinse, four microliters of the MALDI matrix, ACCA(α-cyano-4-hydroxycinnamic acid saturated in 1:2, acetonitrile:1.33%aqueous trifluoroacetic acid), was drawn through the P-10 tips and overthe retained affinity reagent thereby disassociating and enabling laserdesorption/ionization of the myotoxin a and H-myotoxin a. The wholevolume was then immediately driven out of the P-10 tip and placeddirectly onto a mass spectrometer probe tip and allowed to air-drybefore insertion into the vacuum system of the mass spectrometer. Thetime required for sample preparation was approximately one hour.

[0125] Matrix-assisted laser desorption/ionization time-of-flight massspectrometry of the dried material on the mass spectrometer probe tipwas then performed on a linear time-of-flight mass spectrometer. Theinstrument consisted of a 30 kilovolt two-stage acceleration sourcefollowed by a 1.4-meter field free drift region containing a particlewire guide. A frequency-tripled Nd:YAG (355 nm) laser (Lumonics HY 400)was used for desorption/ionization. Ion signals were detected using ahybrid microchannel plate/discrete dynode electron multiplier andrecorded using a 500 MS/s transient recorder (TEKTRONIX TDS 520A)capable of fast signal averaging. The laser irradiance was adjustedduring signal averaging while monitoring the mass spectra on a samplingoscilloscope (TEKTRONIX TDS 310), in order to achieve optimum ion signal(significant signal versus maximum resolution). Time-of-flight spectrumwas generated by signal averaging 50 laser shots into a single spectrumand transferring the data to an IBM compatible personal computer. Datawas processed using the commercially available software, LABCALC(Galactic Industries). The time-of-flight mass spectrum was obtained inthe positive ion mode and externally calibrated with a calibrationequation generated using horse heart cytochrome c (molecular weight (MW)of 12,360 Da).

[0126] The resulting mass spectrum is reproduced in FIG. 2, clearlyshowing a mass spectral signal for myotoxin a at MW=4,822 Da., -A-, anda mass spectral signal for the modified variant H-myotoxin a, atMW=5,242 Da., -B-.

[0127] A 200 μL volume of a second specimen containing 0.002 mg/mL ofwhole venom in human whole blood and 40 nM H-myotoxin like the firstsample above, was mixed with the MALDI matrix, sinapinic acid(acyano-4-hydroxycinnamic acid saturated in 1:2, acetonitrile:1.33%aqueous trifluoroacetic acid), and placed on a mass spectrometric probetip. After drying, the sample was then placed in a time-of-flight massspectrometer and MALDI mass spectrometrically analyzed. The resultingmass spectrum is reproduced in FIG. 3 showing no signals for either ofthe myotoxin a species, rather, the mass spectrum is dominated bysignals due to hemoglobin, i.e., hemoglobin A and B chain signals areobserved at ˜16,000 Da (and doubly-charged at ˜8,000 Da). This exampledemonstrates the necessity of affinity capture and isolation prior toMALDI mass spectrometric analysis to detect myotoxin a.

[0128] The myotoxin a in the first specimen above containing 0.002 mg/mLof whole venom and 40 nM H-myotoxin a, was then quantified using aworking curve strategy. Six preliminary samples which consisted of 50 μLSB, 150 μL tris, and 2 μL×0.02 mg/mL of the H-myotoxin a internalreference (40 nM in each sample) were prepared.

[0129] To each preliminary sample an aliquot of either 5, 10, 20, 30, 40or 50 μL of a purified myotoxin a solution (0.002 mg/mL) was added,resulting in a myotoxin a concentration range of 10 nM to 100 nM. Eachpreliminary sample was then mass spectrometrically immunoassayed as wasthe first specimen except that three 50-laser shot mass spectra wereacquired for each sample rather than one. A specimen containing 2 μLaliquot of C. v. viridis venom at a concentration of 0.2 mg/mL(resulting after dilution in a concentration of 0.002 mg/mL) was treatedin the same manner.

[0130] A six-point normal working curve was generated from the massspectra data of the preliminary samples. The mass spectra of thespecimen and all six preliminary samples were normalized to the signalintensity of the H-myotoxin a. The relative signal intensity of themyotoxin a in the preliminary samples (the average of the three spectra)was then plotted as a function of myotoxin a concentration therebygenerating a six point working curve. FIG. 4 shows the working curvewhich relates the concentration of purified myotoxin a with thenormalized signal intensity of the myotoxin a. Indicated at -5- is thenormalized intensity observed for the specimen. The concentration ofmyotoxin a in the specimen was determined to be 25 nM.

EXAMPLE 2

[0131] Myotoxin a present in a blood sample containing 0.002 mg/mL of C.v. viridis venom was quantified employing the bargraph quantificationstrategy. Introduced into the sample were multiple internal referencespecies of myotoxin a which had been iodinated (at the tyrosineresidues) using the following procedure: Iodobeads (Pierce) were washedtwo times with 0.1 M sodium phosphate at pH 7.0, then incubated for fiveminutes with 0.4 M sodium iodide in a 0.1 M sodium phosphate solution atpH 7.0. An equal volume of myotoxin a, 2 mg/mL in buffer, was added tothe sodium iodide/Iodobead solution. Two Iodobeads were used for eachmilligram of myotoxin a. The mixture was incubated overnight (−10 hours)at room temperature, after which the reaction was stopped by removal ofthe Iodobeads. The modified protein was desalted and stored as describedabove. The reaction resulted in the production of four distinctiodinated species, with each addition incrementing the molecular weightof myotoxin a by 126 Da (the difference between the atomic weight ofiodine and the atomic weight of the displaced proton). Immunoassay andmass spectral procedures were the same as described in Example 1.

[0132] The internal reference species comprising the bargraph werecalibrated against known concentrations of purified myotoxin a (usingthe calibration procedure described in Example 1). Once therelationships between the signal intensities of the myotoxin a and theinternal reference species was established, the concentration ofmyotoxin a present in the sample could be determined, at a glance, bycorrelation with the internal reference peaks of closest intensity. Forthe example shown in FIG. 5, concentrations of 20, 100, 130 and 30 nMare indicated by the signal intensities of the first through fourth MV,-D-, -E-, -F-, and -G-, respectively. As the intensity of the myotoxin asignal -H- is observed to be between that of the first and fourth MV, itis determined that the myotoxin a concentration is in the range of 20 to30 nM.

EXAMPLE 3

[0133]FIG. 6 demonstrates a single-point relative signal strategy forquantifying and detecting myotoxin a in human blood. A human bloodsample containing an unknown concentration of myotoxin a and a 30 nMconcentration of H-myotoxin a was prepared then mass spectrometricallyimmunoassayed according to the protocols of Example 1 above. Theresulting mass spectrum shows mass spectral signals for myotoxin a, -I-,and H-myotoxin a, -J-, at their respective molecular weights. Therelative mass spectrometric responses of myotoxin a and H-myotoxin a maybe determined from the mass spectrum of FIG. 2 in which theconcentrations of both species are known (25 nM myotoxin a, from theworking curve calibration, and 40 nM H-myotoxin a). From thiscalibration it may be determined that equal concentrations of the twoanalytes give a 10% lower signal for H-myotoxin a. The myotoxin a signalis 3.5 times the H-myotoxin a signal for a known 30 nM concentration ofH-myotoxin a. Therefore the myotoxin a concentration is calculated to be(30 nM×3.5/1.1) or 95 nM.

EXAMPLE 4

[0134] A multiplex mass spectrometric immunoassay was performed tosimultaneously detect the presence of myotoxin a and Mojave toxin inhuman blood.

[0135] The protocols of Example 1 above were followed with twoexceptions. First, anti-myotoxin a and anti-Mojave toxin basic subunitimmunoglobulins IgG were immobilized on 6% agarose beads resulting in anaffinity reagent containing antibodies towards both myotoxin a andMojave toxin. Second, the specimens of human blood contained 0.002 mg/mLof the whole venom of the Mojave rattlesnake, Crotalus scutulatusscutulatus (C.s. scutulatus), rather than the venom of C. v. viridis,and the specimens did not contain the internal reference species,H-myotoxin a. The venom of the C.s.scutulatus contains both myotoxin aand Mojave toxin.

[0136] The mass spectrum resulting from the complete mass spectrometricimmunoassay of a specimen is reproduced in FIG. 7, clearly showing amass spectral signal for myotoxin a at MW=4,822 Da., -K-, and Mojavetoxin at MW=14,175 Da., -L-. Also observed in the mass spectrum aresignals due to the hemoglobin present in the blood sample (A- andB-chains at MW of ˜16,000 Da). These did not prove a seriouscomplication to the assay as the toxin signals are clearly observed atresolved values.

[0137] Mere MALDI mass spectrometric analysis of another identicalspecimen without affinity capture and isolation of myotoxin a and Mojavetoxin yielded a mass spectrum with no discernable signals for the toxinssimilar to that shown in FIG. 3.

EXAMPLE 5

[0138] Example 5 demonstrates the detection and quantification of theblood serum protein α-1-acid glycoprotein (hence A1AG) using a massspectrometric immunoassay in which the internal reference species isintrinsic to the biological system was performed. The internal referencespecies in this example is human serum albumin (hence HSA), anotherblood serum protein. A working-curve method was employed. The examplewas performed, to demonstrate principle, in a solution of normal saline.

[0139] Five preliminary samples were prepared containing a constantconcentration of HSA to serve as the internal reference species, andalso containing A1AG in the range of 2.5 nM to 27.5 nM. Each preliminarypreparation was mass spectrometrically immunoassayed with a separateaffinity reagent made of both an immobilized antibody to HSA and animmobilized antibody to A1AG. The resulting mass spectrum from one ofthe preliminary preparations (containing 27.5 nM A1AG) is shown in FIG.8 with the HSA signal at -N- and the A1AG signal at -M-. All resultingmass spectra in this example were normalized to the HSA signal, and thenormalized signal intensities of the A1AG were then used to constructthe working curve depicted in FIG. 9.

[0140] An analytical sample, known to contain 12.5 nM A1AG was massspectrometrically immunoassayed under similar conditions for thepreparations above. The resulting A1AG signal was within thatrepresented on the 5-point working curve of FIG. 9 and is shown at point-O- corresponding to an A1AG concentration of ˜12.5 nM which verifiesthe accuracy of the working curve quantification method

EXAMPLE 6

[0141] The use of mass spectrometric immunoassay and the standardaddition quantification strategy to detect and quantify myotoxin a inhuman blood laced with the venom of the Mojave rattlesnake, C.s.scutuluswas performed. The venom of the Mojave rattlesnake contains bothmyotoxin a and Mojave toxin. The Mojave toxin intrinsic to the specimenis used in this example as an internal reference species to quantifymyotoxin a.

[0142] A venom-laced blood sample was first divided equally amongst fiveseparate containers. Aliquots of purified myotoxin a were added to eachcontainer, resulting in the samples possessing myotoxin a concentrationsof 0, 180, 540, 890 and 1250 nM over that intrinsic to the sample. Allfive samples were then mass spectrometrically immunoassayed according tothe protocols of Example 4 and the resulting mass spectra normalized tothe Mojave toxin signals. FIG. 10 reproduces the results from thestandard addition analysis in which myotoxin a was added at aconcentration of 1250 nM over the intrinsic level. The signals formyotoxin a and Mojave toxin are observed at -P- and -Q-, respectively.

[0143] The normalized myotoxin a signals were then plotted as a functionof the concentration of myotoxin a (added), and the points fit with astraight line as shown in FIG. 11. The standard addition line was thenextrapolated to the baseline (concentration of added myotoxin a). Theintercept point, -R-, indicated the concentration of myotoxin aintrinsic to the specimen to be 190 nM.

EXAMPLE 7

[0144] A multiplex mass spectrometric immunoassay was performed todetect myotoxin a and Mojave toxin in human blood. The myotoxin a wasthen quantified using the bargraph approach and the Mojave toxin wasquantified using a working curve in which the doubly-iodinated speciesserves as an internal reference species. A venom laced blood sample andaffinity reagent were prepared according to the protocols outlined inExample 1 with two exceptions: 1) the venom used to lace a human bloodsample was that of C.s. scutulatus, and 2) two antibodies, anti-myotoxina and anti-Mojave basic subunit, were present in the antibody solutionused to prepare the affinity reagent.

[0145] The mass spectrometric immunoassay proceeded as outlined inExample 1 with the iodinated modified variants of myotoxin a describedin Example 2 introduced into the sample as internal reference species.Calibration of the bargraph was as in Example 2 and the working curve asin Example 6 with the doubly-iodinated species of myotoxin a serving asthe internal reference. The mass spectrum resulting from the analyticalsample containing 0.001 mg/mL C.s. scutulatus is shown in FIG. 12. Massspectral signals are observed at 4,822 Da, -S-, indicating the presenceof myotoxin a, and 14,175 Da, -T-, indicating the presence of Mojavetoxin basic subunit. The iodinated myotoxin a signals are also observedat ˜4,950-5,320 Da, -U-.

[0146] The myotoxin a signal registered between that of the 1st and 4thiodinated species indicating a concentration between 2 and 3 nM. Fromthe working curve it was determined that the Mojave toxin basic subunitconcentration was 15 nM+2.5 nM.

EXAMPLE 8

[0147] The mass spectrometric immunoassay method of determining thepresence of one or more specific antibodies in sera was performed. Themethod involves retrieval of a portion of the general antibodypopulation present in a sample, and use of this portion to construct theaffinity reagent. The affinity reagent is then screened with specificantigens. Antigens are retained by the affinity reagent if the specificantibody is present in the original sample, and will register in themass spectrum (indicating the presence of the specific antibody).

[0148] The specimen was that of blood serum drawn from a rabbitimmunized against the toxin, α-cobratoxin. The affinity reagent wasprepared by mixing 25 μL of protein A supported on 6% agarose beads witha solution containing 50 μL of the serum and 50 μL of 0.01Mtris[Hydroxymethyl] aminomethane, pH 8.2 (tris)) and incubated for twohours (gentle agitation at room temperature). Following incubation, thebeads were washed (3×100 μL tris) and allowed to react at roomtemperature with 50 μL of 0.02 M dimethylpimelimidate dihydrochlorideprepared in 0.2 M triethanolamine (pH 8.2). The reaction was stoppedafter one hour by washing (3×100 μL) with 0.2 M triethanolamine followedby 100 μL of 0.2 M sodium citrate buffer (pH 2.8). The affinity reagentwas finally washed (3×100 μL) with tris and resuspended in 50 μL of thesame buffer.

[0149] A preparation containing multiple antigen species was preparedand MALDI mass spectrometrically analyzed. The resulting mass spectrumin FIG. 13 shows multiple signals corresponding to multiple antigens inthe preparation. The singly charged signal for α-cobratoxin isidentified at -V- at the molecular weight of 7,822 Da.

[0150] The affinity reagent was then incubated with the preparationfollowing similar mass spectrometric immunoassay protocols alreadydescribed to see which of the antigen species from the preparation ofscreening antigens were retained. The resulting mass spectrum is shownin FIG. 14. A signal at the mass-to-charge ratio of α-cobratoxin at7,822 m/z (molecular weight 7,822 Da), -W-, indicates the retention ofα-cobratoxin by the affinity reagent, which in turn indicates thepresence of anti-α-cobratoxin present in the serum from which theaffinity reagent was made.

[0151] From the foregoing, it is readily apparent that new usefulembodiments of the present invention have been herein described andillustrated which fulfills all of the aforestated objectives in aremarkably unexpected fashion. It is of course understood that suchmodifications, alterations and adaptations as may readily occur to theartisan confronted with this disclosure are intended within the spiritof this disclosure which is limited only by the scope of the claimsappended hereto.

Accordingly, what is claimed is:
 1. A method for simultaneouslydetermining whether a specimen contains any of one or more certainantigen species, comprising the steps of: means for capturing andisolating each of said certain antigen species from said specimen, saidcapturing and isolating means involving the use of an affinity reagenthaving a specific affinity for each said certain antigen species; andmeans for detecting the presence of said isolated certain antigenspecies involving the use of a mass spectrometer to determine whethereach said certain antigen species was present in said specimen.
 2. Themethod of claim 1 , further including the steps of: immobilizing atleast one antibody onto a solid substrate to produce said affinityreagent; combining an effective amount of said affinity reagent withsaid specimen until said affinity reagent binds with each of saidcertain antigen species that is present in said specimen to produce apost-combination affinity reagent and an unbound remainder; separatingsaid post-combination affinity reagent from said unbound remainder toform an isolated post-combination affinity reagent; adding a laserdesorption/ionization agent to said isolated post-combination affinityreagent to form a mass spectrometric mixture; and mass spectrometricallyanalyzing said mass spectrometric mixture to produce a mass spectrum,said mass spectrum indicating whether said specimen contained each saidcertain antigen species by exhibiting a mass spectrometric responselocated at the unique mass-to-charge ratio of each said certain antigenspecies.
 3. The method of claim 2 further including the step of adding adisassociation agent to said isolated post-combination affinity reagentprior to said adding laser desorption/ionization agent step.
 4. A methodfor determining how much of one or more certain antigens are present ina specimen, comprising the steps of: adding an internal referencespecies to said specimen where said specimen does not already containone; means for capturing and isolating said certain antigen or antigensand said internal reference species from said specimen, said capturingand isolating means involving the use of an affinity reagent having aspecific affinity for said certain antigen or antigens and said internalreference species; and means for quantifying said certain antigen orantigens, wherein said quantifying means involves mass spectrometricanalysis of said isolated certain antigens and said isolated internalreference species.
 5. The method of claim 4 , further including thesteps of: making at least one standard addition preparation, eachcontaining a known concentration of each said certain antigen or acounterpart of said certain antigen species; dividing said specimen toform a first divided sample and at least one remainder sample; adding aknown amount of each said certain antigen species or said counterpart toeach said remainder sample to produce at least one addition-presentsample wherein the increase in concentration of each said added certainantigen or each said counterpart is known; immobilizing at least oneantibody onto a solid substrate to produce said affinity reagent;combining an effective amount of said affinity reagent with said firstdivided sample to produce an addition-free post-combination affinityreagent and a first unbound remainder; separating said addition-freeaffinity reagent from said first unbound remainder to form an isolatedaddition-free post-combination affinity reagent; adding a laserdesorption/ionization agent to said isolated addition-freepost-combination affinity reagent to form an addition-free massspectrometric mixture; mass spectrometrically analyzing saidaddition-free mass spectrometric mixture to produce an addition-freemass spectrum having an internal reference species mass spectrometricresponse at the unique mass-to-charge ratio of said internal referencespecies, and an addition-free mass spectrometric response at the uniquemass-to-charge ratio of each said certain antigen when said certainantigen is present in said specimen; combining an effective amount ofsaid affinity reagent with each said addition-present sample eachcombining step producing an addition-present post-combination affinityreagent and an unbound remainder; separating each said addition presentpost-combination affinity reagent from each of said unbound remainder toform at least one isolated addition-present post-combination affinityreagent; adding a laser desorption/ionization agent to each saidisolated addition-present post-combination affinity reagent to form anaddition-present mass spectrometric mixture therewith; massspectrometrically analyzing each said addition-present massspectrometric mixture to produce an addition-present mass spectrumhaving an internal reference species mass spectrometric response locatedat the unique mass-to-charge ratio of said internal reference species,and an addition-present mass spectrometric response located at theunique mass-to-charge ratios of each said certain antigen; normalizingeach said addition-present mass spectrum and each said addition-freemass spectrum with the respected said internal reference species massspectrometric response to produce an addition-free normalized antigenmass spectrometric response for the addition-free sample, andaddition-present normalized mass spectrometric response for each saidaddition-present sample; determining a set of changes between saidaddition-free normalized antigen mass spectrometric response and eachsaid addition-present normalized antigen mass spectrometric response foreach said certain antigen by subtracting said addition-free normalizedantigen mass spectrometric response from each said addition-presentnormalized antigen mass spectrometric response for each said certainantigen in each said addition-present sample; determining therelationship between said set of changes and each of the correspondingchanges in concentration for each said standard addition antigen in eachsaid addition-present sample resulting from the addition of saidstandard addition antigen preparation; and quantifying each said certainantigen detected in said specimen using said addition-free andaddition-present normalized mass spectrometric responses and saiddetermined relationship.
 6. The method of claim 5 further including thesteps of adding a disassociation agent to said isolated addition-freeaffinity reagent and to each of said isolated addition-present affinityreagents prior to said adding laser desorption/ionization agent step. 7.The method of claim 4 , further including the steps of: making astandard addition preparation, containing a known concentration of eachsaid certain antigen or a counterpart thereof; immobilizing at least oneantibody onto a solid substrate to produce said affinity reagent;combining an effective amount of said affinity reagent with saidspecimen to produce an addition-free post-combination affinity reagentand a first unbound remainder, said first unbound remainder containingthe majority of each said certain antigen; separating said addition-freepost-combination affinity reagent from said first unbound remainder toform an isolated addition-free post-combination affinity reagent; addinga laser desorption/ionization agent to said isolated addition-freepost-combination affinity reagent to form an addition-free massspectrometric mixture; mass spectrometrically analyzing saidaddition-free mass spectrometric mixture to produce an addition-freemass spectrum having an internal reference species mass spectrometricresponse located at the unique mass-to-charge ratio of said internalreference species, and an addition-free mass spectrometric response atthe unique mass-to-charge ratio of each said certain antigen present insaid specimen; adding a known quantity of said standard additionpreparation to said first unbound remainder to produce anaddition-present first unbound remainder in which the concentration ofeach said certain antigen or said counterpart has been increased by aknown amount; combining an effective amount of said affinity reagentwith said addition-present first unbound remainder to produce aaddition-present post-combination affinity reagent and a second unboundremainder, said second unbound remainder containing the majority of eachsaid certain antigen or said counterpart; separating saidaddition-present post-combination affinity reagent from said secondunbound remainder to form an isolated addition-present post-combinationaffinity reagent; adding a laser desorption/ionization agent to saidisolated addition-present post-combination affinity reagent to form aaddition-present mass spectrometric mixture; mass spectrometricallyanalyzing said addition-present mass spectrometric mixture to produce aaddition-present mass spectrum having an internal reference species massspectrometric response located at the unique mass-to-charge ratio ofsaid internal reference species, and an addition-present massspectrometric response at the unique mass-to-charge ratio of each saidcertain antigen when said certain antigen is present in said specimen;normalizing each said addition-present antigen mass spectrum and eachsaid addition-free mass spectrum with the respective said internalreference species mass spectrometric response to produce anaddition-free normalized antigen mass spectrometric response for theaddition-free sample and an addition-present normalized massspectrometric response for each said addition-present sample;determining a set of changes between each said addition-free normalizedantigen mass spectrometric response and each said addition-presentnormalized antigen mass spectrometric response for each said antigen bysubtracting said addition-free normalized antigen mass spectrometricresponse from each said addition-present normalized antigen massspectrometric response for each said antigen in each saidaddition-present sample; determining the relationship between each saidset of changes and each of the corresponding changes in concentration ofstandard addition in each said addition-present sample resulting fromthe addition of said standard addition antigen preparation; andquantifying each said certain antigen detected in said specimen usingsaid addition-free and addition-present normalized antigen massspectrometric responses and said determined relationship.
 8. The methodof claim 7 further including the step of adding a disassociation agentto each said isolated addition-free post-combination affinity reagent,and to each said isolated addition-present post-combination affinityreagent prior to said adding laser desorption/ionization agent step. 9.The method of claim 4 , further including: immobilizing at least oneantibody onto a solid substrate to produce said affinity reagent;combining an effective amount of said affinity reagent with saidspecimen to produce a post-combination affinity reagent and an unboundremainder; separating said post-combination affinity reagent from saidunbound remainder to form an isolated post-combination affinity reagent;adding a laser desorption/ionization agent to said isolatedpost-combination affinity reagent to form a post-combination affinityreagent mass spectrometric mixture; mass spectrometrically analyzingsaid post-combination affinity reagent mass spectrometric mixture toproduce a post-combination affinity reagent mass spectrum, having a massspectrometric response for said internal reference species located atthe unique mass-to-charge ratio of said internal reference species, andan antigen mass spectrometric response for each said certain antigenspecies located at the unique mass-to-charge ratio of each of saidcertain antigen species when said specimen contained a relevant speciesof said certain antigen species thereby detecting said certain antigenspecies and no mass spectrometric response corresponding to themass-to-charge ratio of said certain antigen species when said specimencontains no detectable amount of said antigen species; making aplurality of preparations, each of said preparations containing apreparation antigen or counterpart thereof which is the same as acertain antigen being sought in said specimen wherein the concentrationof said preparation antigen is varied between said plurality ofpreparations in known amounts; adding sufficient said internal referencespecies to each said preparation so that the concentration of saidinternal reference species is the same or is known for each saidpreparation and said specimen; combining an effective amount of saidaffinity reagent to each of said preparations to produce apost-combination preparation affinity reagent and an unbound preparationremainder; separating each said post-combination preparation affinityreagent from said unbound preparation remainder to form isolatedpost-combination preparation affinity reagents; adding a laserdesorption/ionization agent to each said isolated post-combinationpreparation affinity reagent to form preparation mass spectrometricmixtures; mass spectrometrically analyzing each said preparation massspectrometric mixture, each mass spectrometric analysis to produce apreparation mass spectrum, each of said preparation mass spectracontaining a mass spectrometric response for said internal referencespecies located at the unique mass-to-charge ratio of said internalreference species and a preparation antigen mass spectrometric responsefor each said preparation antigen present in said preparation massspectrometric mixture, said preparation antigen mass spectrometricresponse being located at the unique mass-to-charge ratio of therelevant said preparation antigen; normalizing said specimen massspectrum and each of said preparation mass spectra to the massspectrometric response obtained for each said internal reference speciesto obtain a produce normalized antigen mass spectrometric response and anormalized preparation antigen mass spectrometric response; for eachsaid preparation antigen, determining a mathematical relationshipbetween the relevant said normalized preparation antigen massspectrometric response and the relevant said preparation antigenconcentration; locating on said mathematical relationship the pointswhich respectively pertain to each said normalized antigen mass spectralresponses in said specimen mass spectrum; and determining theconcentration that corresponds to each of said point, therebyquantifying each of said certain antigen species, respectively.
 10. Themethod of claim 9 further including the step of adding a disassociationagent to said isolated post-combination affinity reagent prior to saidadding laser desorption/ionization agent step.
 11. The method of claim 4, wherein a plurality of distinctive internal reference species areadded to said specimen in varied and known concentrations, each saidconcentration being chosen to produce a different mass spectrometricresponse after mass spectrometric immunoassay which is characteristic ofthe mass spectrometric response produced by known concentrations of eachsaid certain antigen after mass spectrometric immunoassay, said methodfurther including: immobilizing at least one antibody onto a solidsubstrate to produce said affinity reagent; combining an effectiveamount of said affinity reagent with said specimen until said affinityreagent binds with each of said internal reference species and each ofsaid certain antigen species that is present in said specimen to producea post-combination affinity reagent and an unbound remainder; separatingsaid post-combination affinity reagent from said unbound remainder toform an isolated post-combination affinity reagent; adding a laserdesorption/ionization agent to said isolated post-combination affinityreagent to form a mass spectrometric mixture; mass spectrometricallyanalyzing said mass spectrometric mixture to produce a mass spectrum,said mass spectrum indicating whether said specimen contained any ofsaid certain antigen species by exhibiting an antigen species massspectrometric response located at the unique mass-to-charge ratio ofeach of said certain antigen species, said mass spectrum also having amass spectrometric response for each of said internal reference species;and interpolating each said antigen species mass spectrometric responseto the internal reference species mass spectrometric responseimmediately above and below in magnitude of each of said antigen speciesmass spectrometric response to quantify each said certain antigenspecies in said specimen.
 12. The method of claim 11 further includingthe step of adding a disassociation agent to said isolatedpost-combination affinity reagent prior to said adding laserdesorption/ionization agent step.
 13. The method of claim 4 , furtherincluding the steps of: making a single reference sample containing aknown concentration of a reference antigen for each said certain antigenwherein each said reference antigen is said certain antigen or acounterpart thereof; adding said internal reference species to saidreference sample such that the ratio of the concentration of saidinternal reference species in said reference sample to the concentrationof said internal reference species in said specimen is known;immobilizing at least one antibody on a solid substrate to produce saidaffinity reagent; combining an effective amount of said affinity reagentwith said specimen to produce a specimen post-combination affinityreagent and an unbound remainder; separating said specimenpost-combination affinity reagent from said unbound remainder to form anisolated specimen post-combination affinity reagent; adding a laserdesorption/ionization agent to said isolated specimen post-combinationaffinity reagent to form a specimen mass spectrometric mixture; massspectrometrically analyzing said specimen mass spectrometric mixture toproduce a specimen mass spectrum having a mass spectrometric responsefor each of said certain antigen species present in said specimenlocated at the respective unique mass-to-charge ratio of each saidcertain antigen species, and a specimen internal reference species massspectrometric response located at the unique mass-to-charge ratio ofsaid internal reference species; combining an effective amount of saidaffinity reagent with said single reference sample to produce areference sample post-combination affinity reagent and a second unboundremainder; separating said post-combination affinity reagent from saidsecond reference sample unbound remainder to form an isolated referencesample affinity reagent; adding a laser desorption/ionization agent tosaid isolated reference sample affinity reagent to form a singlereference sample mass spectrometric mixture; mass spectrometricallyanalyzing said single reference sample mass spectrometric mixture toproduce a single reference sample mass spectrum having a massspectrometric response for each said reference antigen containedtherein; each said mass spectrometric response being located at theunique mass-to-charge ratio of each of said reference antigens, and areference sample internal reference species mass spectrometric responselocated at the unique mass-to-charge ratio of said internal referencespecies; normalizing said specimen mass spectrum and said singlereference sample mass spectrum using each said specimen and saidreference sample internal reference species mass spectrometricresponses; and equating the ratio of each said certain antigennormalized mass spectrometric response and said reference antigennormalized mass spectrometric response to the ratio of the knownconcentration of said reference antigen to the unknown concentration ofsaid certain antigen to determine the concentration os said certainantigen.
 14. The method of claim 13 further including the step of addinga disassociation agent to each of said first and second isolatedaffinity reagent prior to said adding laser desorption/ionization agentstep.
 15. A method for determining whether a specimen contains any ofone or more certain antibody species, comprising the steps of: means forcapturing and isolating each of said certain antibody species from saidspecimen, said capturing and isolating means involving the use of anaffinity reagent having a specific affinity for a different one of saidcertain antibody species; and means for detecting the presence of any ofsaid isolated certain antibody species, involving the use of a massspectrometer, to determine whether each said certain antibody specieswas present in said specimen.
 16. The method of claim 15 , furthercomprising the steps of: immobilizing at least one antigen onto a solidsubstrate to produce said affinity reagent; combining an effectiveamount of said affinity reagent with said specimen to produce apost-combination affinity reagent and an unbound remainder; separatingeach said post-combination affinity reagent from each said unboundremainder to form an isolated post-combination affinity reagent; addinga laser desorption/ionization agent to each said isolatedpost-combination affinity reagent to form a mass spectrometric mixturetherewith; and mass spectrometrically analyzing said mass spectrometricmixture to produce a mass spectrum indicating whether said specimencontained each said certain antibody species by exhibiting a massspectral signal located at the unique mass-to-charge ratio of each saidcertain antibody species.
 17. The method of claim 16 , wherein saidantigen is an allergen.
 18. The method of claim 16 , further includingthe step of adding a disassociation agent to each said isolatedpost-combination affinity reagent prior to said adding laserdesorption/ionization agent step.
 19. The method of claim 18 , whereinsaid antigen is an allergen.
 20. A method for determining the amount ofa certain antibody present in a specimen, comprising the steps of:adding an internal reference species to said specimen when said specimendoes not already contain one; means for capturing and isolating saidcertain antibody and said internal reference species from said specimen,said capturing and isolating means involving the use of an affinityreagent having a specific affinity for said certain antibody and saidinternal reference species; and means for quantifying said certainantibody, wherein said quantifying means involves mass spectrometricanalysis of said isolated certain antibody and said internal referencespecies.
 21. The method of claim 20 , further comprising the steps of:making at least one standard addition preparation, each containing aknown concentration of said certain antibody or a counterpart of saidcertain antibody; dividing said specimen to form a first divided sampleand at least one remainder sample; adding a known amount of said certainantibody or said counterpart of said certain antibody to each saidremainder sample to produce at least one addition-present sample whereinthe increase in concentration of said added certain antibody or saidcounterpart is known; immobilizing at least one affinant onto a solidsubstrate to produce said affinity reagent; combining an effectiveamount of said affinity reagent with said first divided sample toproduce an addition-free post-combination affinity reagent and a firstunbound remainder; separating said addition-free affinity reagent fromsaid first unbound remainder to form an isolated addition-freepost-combination affinity reagent; adding a laser desorption/ionizationagent to said isolated addition-free post-combination affinity reagentto form an addition-free mass spectrometric mixture; massspectrometrically analyzing said addition-free mass spectrometricmixture to produce an addition-free mass spectrum having an internalreference species mass spectrometric response at the uniquemass-to-charge ratio of said internal reference species, and anaddition-free antibody mass spectrometric response at the uniquemass-to-charge ratio of said certain antibody present in said specimen;combining an effective amount of said affinity reagent with each saidaddition-present samples each combination producing an addition-presentpost-combination affinity reagent and an unbound remainder; separatingeach said addition-present post-combination affinity reagent from eachsaid unbound remainder to form at least one isolated addition-presentpost-combination affinity reagent; adding a laser desorption/ionizationagent to each of said isolated addition-present post-combinationaffinity reagent to form at least one addition-present massspectrometric mixture therewith; mass spectrometrically analyzing eachsaid addition-present mass spectrometric mixture to produce anaddition-present mass spectrum having an internal reference species massspectrometric response located at the unique mass-to-charge ratio ofsaid internal reference species, and an addition-present antibody massspectrometric response located at the unique mass-to-charge ratio ofsaid certain antibody; normalizing each said addition-present antibodymass spectrum and each said addition-free antibody mass spectrum withthe respective said internal reference species to produce anaddition-free normalized antibody mass spectrometric response for saidaddition-free sample, and an addition-present normalized antibody massspectrometric response for each said addition-present sample;determining a set of changes between said addition-free normalizedantibody mass spectrometric response and each said addition-presentnormalized antibody mass spectrometric response for said certainantibody species by subtracting said addition-free normalized antibodymass spectrometric response from each said addition-present normalizedantibody mass spectrometric response for each of said addition-presentsample; determining the relationship between said set of changes andeach of the corresponding changes in concentration of said standardaddition antibody in each said addition-present sample resulting fromthe addition of said standard addition antibody preparation; andquantifying said certain antibody detected in said specimen using saidaddition-free and addition-present normalized antibody massspectrometric responses and said determined relationship.
 22. The methodof claim 21 further including the steps of adding a disassociation agentto said isolated addition-free affinity reagent and each of saidisolated post-combination addition-present affinity reagents prior tosaid adding laser desorption/ionization agent step.
 23. The method ofclaim 20 , further including the steps of: making a standard additionpreparation, containing a known concentration of a standard additionantibody, said standard addition antibody being said certain antibody ora counterpart thereof; immobilizing at least one affinant onto a solidsubstrate to produce said affinity reagent; combining an effectiveamount of said affinity reagent with said specimen to produce anaddition-free post-combination affinity reagent and a first unboundremainder, said first unbound remainder containing the majority of saidcertain antibody; separating said addition-free post-combinationaffinity reagent from said first unbound remainder to form an isolatedaddition-free post-combination affinity reagent; adding a laserdesorption/ionization agent to said isolated addition-freepost-combination affinity reagent to form an addition-free massspectrometric mixture; mass spectrometrically analyzing saidaddition-free mass spectrometric mixture to produce an addition-freemass spectrum having an internal reference species mass spectrometricresponse located at the unique mass-to-charge ratio of said internalreference species, and an addition-free antibody mass spectrometricresponse at the unique mass-to-charge ratio of said certain antibodypresent in said specimen; adding a known quantity of said standardaddition preparation to said first unbound remainder sample to producean addition-present first unbound remainder sample in which theconcentration of said standard addition antibody or said counterpart hasbeen increased by a known amount; combining an effective amount of saidaffinity reagent with said addition-present first unbound remaindersample to produce an addition-present post-combination affinity reagentand a second unbound remainder, said second unbound remainder containingthe majority of said certain antibody; separating said addition-presentpost-combination affinity reagent from said second unbound remainder toform a isolated addition-present post-combination affinity reagent;adding a laser desorption/ionization agent to said isolatedaddition-present post-combination affinity reagent to form anaddition-present mass spectrometric mixture therewith; massspectrometrically analyzing said addition-present mass spectrometricmixture to produce an addition-present mass spectrum having an internalreference species mass spectrometric response located at the uniquemass-to-charge ratio of said internal reference species and anaddition-present antibody mass spectrometric response at themass-to-charge ratio of said certain antibody when said certain antibodyis present in said specimen; normalizing each said addition-presentantibody mass spectrum and said addition-free antibody mass spectrumwith the respective said internal reference species mass spectrometricresponse to produce an addition-free normalized antibody massspectrometric response for the addition-free sample and anaddition-present normalized antibody mass spectrometric response foreach said addition-present sample; determining a set of changes betweensaid addition-free normalized antibody mass spectrometric response andeach said addition-present normalized antibody mass spectrometricresponse for said certain antibody by subtracting said addition-freenormalized antibody mass spectrometric response from each saidaddition-present normalized antibody mass spectrometric response forsaid certain antigen in each said addition-present sample; determiningthe relationship between said set of changes and each of thecorresponding changes in concentration of standard addition in each saidaddition-present sample resulting from the addition of said standardaddition antibody preparation; and quantifying said certain antibodydetected in said specimen using said addition-free and addition-presentnormalized antibody mass spectrometric responses and said determinedrelationship.
 24. The method of claim 23 further including the step ofadding a disassociation agent to said isolated addition-freepost-combination affinity reagent, and each said isolatedaddition-present post-combination affinity reagent prior to said addinglaser desorption/ionization agent step.
 25. The method of claim 20 ,further comprising the steps of: immobilizing at least one affinant ontoa solid substrate to produce said affinity reagent; combining aneffective amount of said affinity reagent with said specimen to producea post-combination affinity reagent and an unbound remainder; separatingsaid post-combination affinity reagent from said unbound remainder toform an isolated post-combination affinity reagent; adding a laserdesorption/ionization agent to said isolated post-combination affinityreagent to form a post-combination affinity reagent mass spectrometricmixture; mass spectrometrically analyzing said post-combination affinityreagent mass spectrometric mixture to produce a post-combinationaffinity reagent mass spectrum, having a mass spectrometric response forsaid internal reference species located at the unique mass-to-chargeratio of said internal reference species and a mass spectrometricresponse for said certain antibody located at the mass-to-charge ratioof said certain antibody and no mass spectrometric responsecorresponding to the mass-to-charge ratio of said certain antigenspecies when said specimen contains no detectable amount of saidantibody species; making a plurality of preparations containing apreparation antibody which is said certain antibody being quantified insaid specimen or a counterpart thereof, wherein said preparationantibody concentration varies in known fashion between saidpreparations; adding sufficient said internal reference species to saidpreparations so that the concentration of said internal referencespecies is the same or is known for each of said preparations and saidspecimen; combining an effective amount of said affinity reagent witheach of said preparations each combination to produce a post-combinationpreparation affinity reagent and an unbound preparation remainder;separating each said post-combination preparation affinity reagent fromsaid unbound preparation remainder to form an isolated post-combinationpreparation affinity reagent; adding a laser desorption/ionization agentto each said isolated post-combination preparation affinity reagents toform a preparation mass spectrometric mixtures; mass spectrometricallyanalyzing each said preparation mass spectrometric mixture, each massspectrometric analysis to produce a preparation mass spectrum, each ofsaid preparation mass spectra having a mass spectrometric response forsaid internal reference species located at the mass-to-charge ratio ofsaid internal reference species and a mass spectrometric response forsaid preparation antibody located at the mass-to-charge ratio of saidpreparation antibody; normalizing said specimen mass spectrum and eachof said preparation mass spectra to the mass spectrometric responsesobtained for each said internal reference species obtain a normalizedcertain antibody mass spectrometric response and normalized preparationantibody mass spectrometric response; determining the mathematicalrelationship between said normalized preparation antibody massspectrometric responses and said preparation antibody concentrations;locating a point on said mathematical relationship which appertains tosaid normalized certain antibody mass spectral response; and determiningthe concentration that corresponds to said point, thereby quantifyingsaid certain antibody.
 26. The method of claim 25 further including thestep of adding a disassociation agent to said isolated post-combinationaffinity reagent prior to said adding laser desorption/ionization agentstep.
 27. The method of claim 20 , further including the steps of:preparing a single reference sample containing a known concentration ofa reference antibody, wherein said reference antibody is said certainantibody or a counterpart thereof; immobilizing at least one affinant ona solid substrate to produce said affinity reagent; combining aneffective amount of said affinity reagent with said specimen to producea specimen post-combination affinity reagent and a first unboundremainder; separating said specimen post-combination affinity reagentfrom said first unbound remainder to form a first isolated specimenpost-combination affinity reagent; adding a laser desorption/ionizationagent to said first isolated specimen post-combination affinity reagentto form a specimen mass spectrometric mixture; mass spectrometricallyanalyzing said specimen mass spectrometric mixture to produce a specimenmass spectrum having a mass spectrometric response for said certainantibody at the mass-to-charge ratio of said certain antibody, and alsohaving a specimen internal reference species mass spectrometric responselocated at the mass-to-charge ratio of said internal reference species;combining an effective amount of said affinity reagent with said singlereference sample to produce a reference sample post-combination affinityreagent and a second unbound remainder; separating said reference samplepost-combination affinity reagent from said second unbound remainder toform an isolated reference sample post-combination affinity reagent;adding a laser desorption/ionization agent to said isolated referencesample post-combination affinity reagent to form a single referencesample mass spectrometric mixture; mass spectrometrically analyzing saidsingle reference sample mass spectrometric mixture to produce a singlereference sample mass spectrum having a mass spectrometric response forsaid reference antibody located at the unique mass-to-charge ratio ofsaid reference antibody, and a second internal reference species massspectrometric response located at the mass-to-charge ratio of saidinternal reference species; normalizing said specimen mass spectrum andsaid single reference sample mass spectrum using said first and secondinternal reference species mass spectrometric responses; and equatingthe ratio of the magnitudes of said certain antibody mass spectrometricresponse and said reference antibody mass spectrometric response to theratio of the known concentration of said counterpart antibody and theunknown concentration of said certain antibody to determine theconcentration of said certain antibody.
 28. The method of claim 27further comprising the step of adding a disassociation agent to each ofsaid first and second isolated affinity reagent prior to said addinglaser desorption/ionization agent step.
 29. A method of simultaneouslydetermining whether an antibody population contained in a specimencontains one or more certain antibody species, comprising the steps of:combining said specimen with a solid substrate having a non-specificaffinity for antibodies to immobilize said antibody population onto saidsolid substrate and produce an affinity reagent; placing said affinityreagent into a preparation containing at least one known antigen, eachsaid known antigen having a specific affinity for one of said certainantibodies to produce a post-combination affinity reagent and a screenedpreparation; separating said post-combination affinity reagent from saidscreened preparation to form an isolated post-combination affinityreagent; adding a laser desorption/ionization agent to said isolatedpost-combination affinity reagent to form a mass spectrometric mixture;and mass spectrometrically analyzing said mass spectrometric mixture toproduce a mass spectrum, said mass spectrum indicating whether saidantiserum contained each of said certain antibody species by exhibitinga mass spectrometric response located at the unique mass-to-charge ratioof the relevant said known antigen which has a specific affinity forthat said certain antibody species.
 30. The method of claim 29 furtherincluding the step of adding a disassociation agent to said isolatedpost-combination affinity reagent prior to said adding laserdesorption/ionization agent step.